Phospholipid drug analogs

ABSTRACT

Provided in some embodiments are compositions comprising a compound having a structure according to Formula A or Formula B: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt, tautomer or hydrate thereof, where X 2  is a bond or linker, X 3  is bond or —PO 4 —, and X 1 , R 1 , R 2 , R 3 , and n are described herein. Also provided in some embodiments are methods for making and using such compounds and compositions.

RELATED PATENT APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/097,838 filed Apr. 29, 2011, entitled PHOSPHOLIPID DRUGANALOGS naming Roberto Maj, Franco Pattarino, Emanuela Mura and AlcideBarberis as inventors, and designated by attorney docket no.TEL-1500-UT, which claims the benefit of U.S. provisional patentapplication No. 61/330,151 filed Apr. 30, 2010, entitled PHOSPHOLIPIDDRUG ANALOGS, naming Alcide Barberis et al. as inventors, and designatedby attorney docket no. TEL-1500-PV. This application claims the benefitof U.S. provisional patent application No. 61/708,513 filed Oct. 1,2012, entitled PHOSPHOLIPID DRUG ANALOGS, naming Roberto Maj, FrancoPattarino, Emanuela Mura and Alcide Barberis as inventors, anddesignated by attorney docket no. TEL-1502-PV. Each of the foregoingpatent applications is incorporated herein by reference in its entirety,including all text, tables and drawings.

FIELD

The technology relates in part to phospholipid drug analogs, and methodsfor manufacturing and using the same.

BACKGROUND

A pharmacophore often is a molecule that can exert a therapeutic effectin a subject. For example, a pharmacophore sometimes can exert ananti-cell proliferation effect, which can be useful for treating cellproliferation conditions such as cancer. A pharmacophore sometimes canstimulate the immune system in a subject, and thereby can generate orenhance an immune response against a particular antigen.

A pharmacophore can be conjugated (e.g., linked) to a phospholipid, orphospholipid-like molecule, in a phospholipid drug analog. Aphospholipid, or phospholipid-like component, can impart a function tothe analog that differs from the action of the unconjugatedpharmacophore.

SUMMARY

Provided in some embodiments are compositions comprising a compoundhaving a structure according to Formula A or Formula B:

or a pharmaceutically acceptable salt, tautomer or hydrate thereof,where:

-   -   X¹ is —O—, —S—, or —NR^(a)—;    -   R^(a) is hydrogen, C1-C10 alkyl, or substituted C1-C10 alkyl, or        R^(a) and R¹ taken together with the nitrogen atom can form a        heterocyclic ring or a substituted heterocyclic ring, where the        substituents on the alkyl or heterocyclic groups are hydroxy,        C1-C10 alkyl, hydroxyl C1-C10 alkenyl, C1-C6 alkoxy, C3-C6        cycloalkyl, C1-C6 alkoxy C1-C6 alkylene, amino, cyano, halogen        or aryl;    -   R¹ is hydrogen, C1-C10 alkyl, substituted C1-C10 alkyl, C1-C10        alkoxy, substituted C1-C10 alkoxy, C3-C9 cycloalkyl, substituted        C3-C9 cycloalkyl, C5-C10 aryl, substituted C5-C10 aryl, C5-C9        heterocyclic, substituted C5-C9 heterocyclic, C1-C6 alkanoyl,        Het, Het C1-C6 alkyl, or C1-C6 alkoxycarbonyl, where the        substituents on the alkyl, cycloalkyl, alkanoyl,        alkcoxycarbonyl, Het, aryl or heterocyclic groups are hydroxyl,        C1-C10 alkyl, hydroxyl C1-C10 alkylene, C1-C6 alkoxy, C3-C9        cycloalkyl, C5-C9 heterocyclic, C16 alkoxy C16 alkenyl, amino,        cyano, halogen or aryl;    -   each R² independently is hydrogen, —OH, C1-C6 alkyl, substituted        C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, —C(O)—C1-C6        alkyl (alkanoyl), substituted —C(O)—C1-C6 alkyl, —C(O)—C6-C10        aryl (aroyl), substituted —C(O)—C6-C10 aryl, —C(O)OH (carboxyl),        —C(O)O—C1-C6 alkyl (alkoxycarbonyl), substituted —C(O)O—C1-C6        alkyl, —NR^(a)R^(b), —C(O)NR^(b)R^(c) (carbamoyl), substituted        C(O)NR^(b)R^(c), C5-C9 cyclic, substituted C5-C9 cyclic, C5-C9        heterocyclic, substituted C5-C9 heterocyclic, halo, nitro, or        cyano, where the substituents on the alkyl, cyclic, aryl or        heterocyclic groups are hydroxy, C1-C10 alkyl, hydroxyl C1-C10        alkylene, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 alkoxy C1-C6        alkylene, amino, cyano, halogen or aryl;    -   each R^(b) and R^(c) independently is hydrogen, C1-C10 alkyl,        substituted C1-C10 alkyl, C1-C10 alkoxy, substituted C1-C10        alkoxy, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C10        aryl, substituted C5-C10 aryl, C5-C9 heterocyclic, substituted        C5-C9 heterocyclic, C1-C6 alkanoyl, Het, Het C1-C6 alkyl, or        C1-C6 alkoxycarbonyl, where the substituents on the alkyl,        cycloalkyl, alkanoyl, alkcoxycarbonyl, Het, aryl or heterocyclic        groups are hydroxyl, C1-C10 alkyl, hydroxyl C1-C10 alkylene,        C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C16 alkoxy        C16 alkenyl, amino, cyano, halogen or aryl;    -   X² is a bond or a linking group; n is 0, 1, 2, 3 or 4; and    -   X³ is a bond or a —PO₄—;    -   R³ is a C1-C6 alkyl substituted with —OC(O)—R^(d) and        —OC(O)—R^(e); C1-C6 alkyl substituted with —OC(O)—R^(d),        —OC(O)—R^(e), and one or more further substituents; C1-C6        alkenyl substituted with —OC(O)—R^(d) and —OC(O)—R^(e); or C1-C6        alkenyl substituted with —OC(O)—R^(d), —OC(O)—R^(e), and one or        more further substituents; where the one or more further        substituents independently are hydroxyl, C1-C10 alkyl, hydroxyl        C1-C10 alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9        heterocyclic, C16 alkoxy C16 alkylene, amino, cyano, halogen or        aryl;    -   each R^(d) and R^(e) independently is a linear C10 to C16 alkyl        or linear C10 to C16 alkyl substituted with one or more of        hydroxyl, C1-C10 alkyl, hydroxyl C1-C10 alkylene, C1-C6 alkoxy,        C3-C6 cycloalkyl, C16 alkoxy C16 alkylene, amino, cyano, epoxy,        halogen or aryl.

In certain embodiments, R^(d) and R^(e) independently are a linear andsaturated C10, C11, C12, C13, C14, C15 or C16 alkyl, and in someembodiments R^(d) and R^(e) are the same or different. Non-limitingexamples of —OC(O)—R^(d) and —OC(O)—R^(e) independently includen-decanoyl (C10, —OC(O)—(CH₂)₈CH₃), n-dodecanoyl (C12, lauroyl,—OC(O)—(CH₂)₁₀CH₃), n-tetradecanoyl (C14, myristoyl, —OC(O)—(CH₂)₁₂CH₃),and n-hexadecanoyl (C16, palmitoyl, —OC(O)—(CH₂)₁₄CH₃). R^(d) and R^(e),in some embodiments, independently are a linear and saturated C11 to C15alkyl. Sometimes R^(d) and R^(e) independently are a linear andsaturated C10, C11, C12, C13, C14, C15 or C16 alkyl. In someembodiments, R^(d) and R^(e) independently are a linear and saturatedC10-C16 alkyl (e.g., C10, C11, C12, C13, C14, C15 or C16 alkyl), or alinear C10-C16 alkyl substituted with one or more of hydroxyl, C1-C10alkyl, hydroxyl C1-C10 alkylene, C1-C6 alkoxy, C3-C6 cycloalkyl, C16alkoxy C16 alkylene, amino, cyano, epoxy, halogen or aryl. In someembodiments, R^(d) and R^(e) are linear and saturated (for example, SC12and SC14), and in some embodiments, R^(d) and R^(e) are linear andnonsaturated (for example, Compound A). Each linear C10, C11, C12, C13,C14, C15 or C16 alkyl or substituted variant thereof sometimes is fullysaturated and contains no unsaturation (i.e., double bond). Each linearC10, C11, C12, C13, C14, C15 or C16 alkyl is an alkyl moiety having 10,11, 12, 13, 14, 15 or 16 carbon atoms, respectively.

In specific embodiments, each R^(d) and R^(e) is a saturated and linearC1-2 alkyl. In some embodiments, R^(d) and R^(e) are not substituted byan epoxy moiety, and sometimes R^(d) and R^(e) are not substituted by ahydroxyl moiety. In specific embodiments, R^(d) and R^(e) are notsubstituted by an epoxy moiety or a hydroxyl moiety. In variousembodiments, R^(d) and R^(e) include no double bond (e.g., nounsaturation).

In some embodiments, —X²—X³—R³ together form a structure according toFormula C:

In certain embodiments —X²—X³—R³ taken together form a structureaccording to Formula D:

In some embodiments, X¹ is O, and sometimes R¹ is a C1-C10 alkylsubstituted with a C16 alkoxy. In certain embodiments n is 0 and X² is—C(O)NH—(CH₂)₂—. Sometimes R³ is a C3 alkyl substituted with—OC(O)—R^(d) and —OC(O)—R^(e), and in certain embodiments, the R³ C3alkyl is substituted with —OC(O)—R^(d) at position 3 and —OC(O)—R^(e) atposition 2 of the C3 alkyl (e.g., see Formula C, where the —PO₄— moietyis linked to position 1 of the C3 alkyl, the —OC(O)—R^(e) moiety is atposition 2 and the —OC(O)—R^(d) moiety is at position 3). In specificembodiments, X¹ is O, R¹ is —(CH₂)₂—OCH₃, n is 0, X² is —C(O)NH—(CH₂)₂—,X³ is —PO₄—, R³ is a C3 alkyl substituted with —OC(O)—R^(d) and—OC(O)—R^(e). In some embodiments, R^(d) and R^(e) are not substitutedby an epoxy moiety and/or are not substituted by a hydroxyl moiety.

In some embodiments, the benzene ring in Formula A or Formula B isreplaced with a non-aromatic ring, a heterocyclic non-aromatic ring, ora heterocyclic aromatic ring. Examples of these rings include, forexample, those listed herein. Non-limiting examples of non-aromaticrings include any 5- or 6-membered cycloalkyl or heterocyclic moieties.Non-limiting examples of heterocyclic non-aromatic rings includepiperidine and piperazine. Non-limiting examples of heterocyclicaromatic rings include, for example, pyridine, pyrazine, pyrimidine,pyridazine, and triazine.

In certain embodiments, a composition comprises a liposome. Acomposition in some embodiments comprises an antigen.

Provided also in some embodiments are immunostimulatory compositionscomprising a compound having a structure described herein. In certainembodiments, the compound functions as an adjuvant, and sometimes animmunostimulatory composition comprises an antigen (e.g., thecomposition functions as a vaccine). An immunostimulatory composition insome embodiments comprises a vaccine, and in some embodiments animmunostimulatory composition is a primary vaccine or combinationvaccine.

Also provided are methods of treating a condition in a subject whichcomprises administering a composition described herein to a subject inneed thereof in an amount effective to treat the condition. In someembodiments the subject is a mammal, such as a human, for example. Insome embodiments the condition is a cancer condition, such as a bladdercancer condition, for example. In some embodiments the composition isadministered by intravesical instillation or by topical delivery to abladder. In some embodiments, the condition is a microbial infection. Insome embodiments, the condition is a skin precancerous or cancerouscondition.

Also provided are methods of inducing an immune response comprisingadministering to a subject a compound having a structure providedherein. By inducing an immune response is meant inducing an immuneresponse to a specific antigen, or inducing a general immune response(in the absence of a specific antigen). In one embodiment, the compoundacts as an adjuvant and so is associated with a specific, not a generalimmune response. In one embodiment, the compound acts as a generalimmune stimulator. In one embodiment, the method includes administeringto a mammal in need thereof an amount of an antigen and a compoundhaving a structure provided herein effective to prevent, inhibit ortreat disorders, including but not limited to bladder cancer or skincancer. Thus, in certain embodiments, the immune response is anantigen-specific immune response. In some embodiments, the immuneresponse is an antibody response, which sometimes is, for example, aIgG1 or a IgG2a antibody response. In some embodiments, the antigen is amicrobial antigen, for example, a Malaria antigen may be administered,in some embodiments, the antigen is an E. coli antigen. The antigen andthe compound sometimes are in one composition, and in some embodimentsthe antigen and the compound are in different compositions. The compoundand/or antigen in certain embodiments is in association with a liposome.The antigen and the compound may be administered at the same time, or atdifferent times. In some embodiments, the antigen is administered beforethe compound, in other embodiments, the antigen is administered at thesame time as the compound, in other embodiments, the antigen isadministered after the compound. In some embodiments, the immuneresponse is an antigen specific immune response. In certain embodiments,the immune response is an antibody response, which sometimes is an IgG2aantibody response.

In some embodiments the subject is a mammal, such as a human, forexample. In certain embodiments, the compound is administered to thebladder, such as by intra-vesical instillation/topical delivery, innon-limiting embodiments.

In some embodiments, the compound is administered to the skin, forexample, by topical delivery. Also provided in certain embodiments aremethods for treating a condition in a subject, which compriseadministering a composition described herein to a subject in needthereof in an amount effective to treat the condition. Provided also insome embodiments are methods for treating a condition in a subject,which comprise administering an immunostimulatory composition describedherein to a subject in need thereof in an amount effective to treat thecondition. The subject sometimes is a mammal, and can be a human incertain embodiments. The condition sometimes is a cancer condition, andthe condition can be a microbial infection. In specific embodiments, thecondition is a bladder cancer condition, and the composition can beadministered by intravesical instillation/topical delivery to thebladder, in certain embodiments. In some embodiments, the cancer is askin cancer, and the compounds can be administered locally and/ortopically, for example, by topical delivery to the skin, in a cream,ointment, gel, lotion or other appropriate vehicle. Skin precancerousconditions and skin cancers that may be treated include, for example,actinic keratosis (AK), basal cell carcinoma (BCC), squamous cellcarcinoma (SCC), melanoma and non-melanoma skin cancer.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 illustrates cytokine production for compounds in Raw264.7 mousemacrophage cell line studies.

FIG. 2 illustrates cytokine production for compounds in Raw264.7 mousemacrophage cell line studies.

FIGS. 3A and 3B show survival data for Raw264.7 mouse macrophage cellline studies.

FIG. 4 is a graph of IL-6 production for compounds in PBMC studies forDonor 1.

FIG. 5 is a graph of IL-6 production for compounds in PBMC studies forDonor 2.

FIG. 6 is a graph of IL-6 production for compounds in PBMC studies forDonor 3.

FIG. 7 is a graph of TNF-alpha production for compounds in PBMC studiesfor Donor 1.

FIG. 8 is a graph of TNF-alpha production for compounds in PBMC studiesfor Donor 2.

FIG. 9 is a graph of TNF-alpha production for compounds in PBMC studiesfor Donor 3.

FIGS. 10A-10G provide survival data for PBMC studies.

FIG. 11 shows structures and molecular weights of phospholipid analogsSC8, SC12 and SC18.

FIG. 12 shows MFI values for CD40 expression on double positiveHLA-DR+/CD20+B cells after 24 hours incubation with test reagents asindicated, performed on whole blood from three donors.

FIGS. 13A and 13B illustrate MFI values for CD80, CD86, and CCR7expression in HLA-DR+/CD11c+/CD123-mDCs after 24 hour incubation withtest reagents as indicated, performed on whole blood from Donor 2.

FIGS. 14A and 14B show MFI values for CD80, CD86, and CCR7 expression inHLA-DR+/CD11c+/CD123-mDCs after 24 hour incubation with test reagents asindicated, performed on whole blood from Donor 3.

FIGS. 15A and 15B illustrate MFI values for CD80, CD86, and CCR7expression in HLA-DR+/CD11c+/CD123-mDCs after 24 hour incubation withtest reagents as indicated, performed on whole blood from Donor 1.

FIGS. 16A and 16B show MFI values for CD80, CD86, and CCR7 expression inHLA-DR+/CD11c−/CD123+pDCs after 24 hour incubation with test reagents asindicated, performed on whole blood from three donors (D1-D3).

FIGS. 17A-17D are a collection of bar charts of the cytotoxic effects ofSC12 and Imiquimod on cells. Cutaneous SCC cell lines were used tocontinuously monitor of electric conductance in microtiter wells(E-plates, Roche), which correspond to the cell numbers. TMX indicatesSC12 in the charts.

FIG. 18 is a collection of photographs of cells contacted with SC12 orImiquimod Similar morphological changes were induced by SC12 andImiquimod. At day 3, cell detachment, morphological changes andinhibition of proliferation can be observed in SCC cells treated witheither SC12 or Imiquimod.

FIGS. 19A and 19B show the development of IgG titers against the M.ulcerans antigen, (upper: compound A, lower, SC12).

FIGS. 20A and 20B provide an example of a synthetic scheme for synthesisof compound A and SC12.

FIGS. 21A and 21B provide an example of a synthetic scheme for synthesisof compound A and SC12.

FIGS. 22A and 22B provide an example of a synthetic scheme for synthesisof compound A and SC12.

FIGS. 23A and 23B provide an example of a synthetic scheme for synthesisof compound A and SC12.

FIGS. 24A, 24B and 24C show SC14 and SC12 inducing TNF-alpha releasefrom human PBMC. FIGS. 24A, 24B and 24C represent PBMCs isolated fromhuman donors 1, 2 and 3 respectively.

FIGS. 25A, 25B and 25C show SC14 and SC12 inducing IL-6 release fromhuman PBMC. FIGS. 25A, 25B and 25C represent PBMCs isolated from humandonors 1, 2 and 3 respectively.

DETAILED DESCRIPTION

Compositions provided herein may be useful for treating certainconditions, such as cell proliferation conditions, for example. Avariety of cell proliferation conditions exist, superficial bladdercancer being an example of one type. Compositions provided herein alsomay serve as a vaccine, and compounds described may facilitate an immuneresponse and provide adjuvant activity.

Compositions provided include phospholipid analogs in certainembodiments. A phospholipid analog often includes a pharmocophoreportion and a phospholipid, or phospholipid-like, portion conjugated tothe pharmacophore portion via a linker.

Without being limited by theory, compositions described herein maymodulate an activity of one or more toll-like receptors (e.g., theconjugates are agonists, antagonists, or both). The term “toll-likereceptor” (TLR) refers to a member of a family of receptors that bind topathogen-associated molecular patterns (PAMPs) and facilitate an immuneresponse in a mammal. Ten mammalian TLRs are known, e.g., TLR1-10. Theterm “toll-like receptor agonist” (TLR agonist) refers to a moleculethat interacts with a TLR and stimulates the activity of the receptor.Synthetic TLR agonists are chemical compounds that are designed tointeract with a TLR and stimulate the activity of the receptor. The term“toll-like receptor antagonist” (TLR antagonist) refers to a moleculethat interacts with a TLR and inhibits or neutralizes the signalingactivity of the receptor. Synthetic TLR antagonists are chemicalcompounds designed to interact with a TLR and interfere with theactivity of the receptor. Agonists and/or antagonists of a TLR sometimesmodulate the activity of a TLR-7, TLR-3 or TLR-9. Local activation of aTLR may disrupt cancer cell-matrix interactions required for growth andsurvival of malignant cells and may induce apoptosis.

Also, without being limited by theory, compounds provided herein can becharacterized as having an advantageous stability. For example, certaincompounds described herein can be characterized as having anadvantageous chemical stability and/or metabolic stability underphysiologic conditions.

Compounds

As used herein, the terms “alkyl,” “alkenyl” and “alkynyl” includestraight-chain (referred to herein as “linear”), branched-chain(referred to herein as “non-linear”), cyclic monovalent hydrocarbylradicals, and combinations of these, which contain only C and H atomswhen they are unsubstituted. Non-limiting examples of alkyl moietiesinclude methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl,2-propenyl, 3-butynyl, and the like. The total number of carbon atoms ineach such group is sometimes described herein, e.g., when the group cancontain up to ten carbon atoms it can be represented as 1-10C or asC1-C10 or C1-10. When heteroatoms (N, O and S typically) are allowed toreplace carbon atoms as in heteroalkyl groups, for example, the numbersdescribing the group, though still written as e.g. C1-C6, represent thesum of the number of carbon atoms in the group plus the number of suchheteroatoms that are included as replacements for carbon atoms in thebackbone of the ring or chain being described. An alkyl that containsonly C and H atoms and is unsubstituted sometimes is referred to as“saturated.” An alkenyl or alkynyl generally is “unsaturated” as itcontains one or more double bonds or triple bonds, respectively. Analkenyl can include any number of double bonds, such as 1, 2, 3, 4 or 5double bonds, for example. An alkynyl can include any number of triplebonds, such as 1, 2, 3, 4 or 5 triple bonds, for example.

Alkyl, alkenyl and alkynyl substituents sometimes contain 1-10C (alkyl)or 2-10C (alkenyl or alkynyl). They can contain 1-8C (alkyl) or 2-8C(alkenyl or alkynyl) in some embodiments. Sometimes they contain 1-4C(alkyl) or 2-4C (alkenyl or alkynyl). A single group can include morethan one type of multiple bond, or more than one multiple bond. Suchgroups are included within the definition of the term “alkenyl” whenthey contain at least one carbon-carbon double bond, and are includedwithin the term “alkynyl” when they contain at least one carbon-carbontriple bond.

Alkyl, alkenyl and alkynyl groups often are optionally substituted tothe extent that such substitution can be synthesized and can exist.Typical substituents include, but are not limited to, halo, ═O, ═N—CN,═N—OR, ═NR, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR, NRCOR,CN, COOR, CONR₂, OOCR, COR, and NO₂, wherein each R is independently H,C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10aryl, or C5-C10 heteroaryl, and each R is optionally substituted withhalo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′₂, SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′,NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′, CONR′₂, OOCR′, COR′, and NO₂,wherein each R′ is independently H, C1-C8 alkyl, C2-C8 heteroalkyl,C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl. Alkyl,alkenyl and alkynyl groups can also be substituted by C1-C8 acyl, C2-C8heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which can besubstituted by the substituents that are appropriate for the particulargroup.

“Acetylene” substituents are 2-10C alkynyl groups that are optionallysubstituted, and are of the formula —C≡C—Ri, wherein Ri is H or C1-C8alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl,C5-C10 heteroaryl, C7-C1-2 arylalkyl, or C6-C1-2 heteroarylalkyl, andeach Ri group is optionally substituted with one or more substituentsselected from halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′2, SR′, SO₂R′,SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′, CONR′₂,OOCR′, COR′, and NO₂, wherein each R′ is independently H, C1-C6 alkyl,C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C10 aryl, C5-C10heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each of which isoptionally substituted with one or more groups selected from halo, C1-C4alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino,and ═O; and where two R′ can be linked to form a 3-7 membered ringoptionally containing up to three heteroatoms selected from N, O and S.In some embodiments, Ri of —C≡C—Ri is H or Me.

“Heteroalkyl”, “heteroalkenyl”, and “heteroalkynyl” and the like aredefined similarly to the corresponding hydrocarbyl (alkyl, alkenyl andalkynyl) groups, but the ‘hetero’ terms refer to groups that contain oneto three O, S or N heteroatoms or combinations thereof within thebackbone residue; thus at least one carbon atom of a correspondingalkyl, alkenyl, or alkynyl group is replaced by one of the specifiedheteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynylgroup. The typical and preferred sizes for heteroforms of alkyl, alkenyland alkynyl groups are generally the same as for the correspondinghydrocarbyl groups, and the substituents that may be present on theheteroforms are the same as those described above for the hydrocarbylgroups. For reasons of chemical stability, it is also understood that,unless otherwise specified, such groups do not include more than twocontiguous heteroatoms except where an oxo group is present on N or S asin a nitro or sulfonyl group.

While “alkyl” as used herein includes cycloalkyl and cycloalkylalkylgroups, the term “cycloalkyl” may be used herein to describe acarbocyclic non-aromatic group that is connected via a ring carbon atom,and “cycloalkylalkyl” may be used to describe a carbocyclic non-aromaticgroup that is connected to the molecule through an alkyl linker.Similarly, “heterocyclyl” may be used to describe a non-aromatic cyclicgroup that contains at least one heteroatom as a ring member and that isconnected to the molecule via a ring atom, which may be C or N; and“heterocyclylalkyl” may be used to describe such a group that isconnected to another molecule through a linker. The sizes andsubstituents that are suitable for the cycloalkyl, cycloalkylalkyl,heterocyclyl, and heterocyclylalkyl groups are the same as thosedescribed above for alkyl groups. As used herein, these terms alsoinclude rings that contain a double bond or two, as long as the ring isnot aromatic.

As used herein, “acyl” encompasses groups comprising an alkyl, alkenyl,alkynyl, aryl or arylalkyl radical attached at one of the two availablevalence positions of a carbonyl carbon atom, and heteroacyl refers tothe corresponding groups wherein at least one carbon other than thecarbonyl carbon has been replaced by a heteroatom chosen from N, O andS. Thus heteroacyl includes, for example, —C(═O)OR and —C(═O)NR₂ as wellas —C(═O)-heteroaryl.

Acyl and heteroacyl groups are bonded to any group or molecule to whichthey are attached through the open valence of the carbonyl carbon atom.Typically, they are C1-C8 acyl groups, which include formyl, acetyl,pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, which includemethoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups,aryl groups, and heteroforms of such groups that comprise an acyl orheteroacyl group can be substituted with the substituents describedherein as generally suitable substituents for each of the correspondingcomponent of the acyl or heteroacyl group.

“Aromatic” moiety or “aryl” moiety refers to a monocyclic or fusedbicyclic moiety having the well-known characteristics of aromaticity;examples include phenyl and naphthyl. Similarly, “heteroaromatic” and“heteroaryl” refer to such monocyclic or fused bicyclic ring systemswhich contain as ring members one or more heteroatoms selected from O, Sand N. The inclusion of a heteroatom permits aromaticity in 5 memberedrings as well as 6 membered rings. Typical heteroaromatic systemsinclude monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl,pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl,and imidazolyl and the fused bicyclic moieties formed by fusing one ofthese monocyclic groups with a phenyl ring or with any of theheteroaromatic monocyclic groups to form a C8-C10 bicyclic group such asindolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl,quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl,quinoxalinyl, cinnolinyl, and the like. Any monocyclic or fused ringbicyclic system which has the characteristics of aromaticity in terms ofelectron distribution throughout the ring system is included in thisdefinition. It also includes bicyclic groups where at least the ringwhich is directly attached to the remainder of the molecule has thecharacteristics of aromaticity. Typically, the ring systems contain 5-12ring member atoms. The monocyclic heteroaryls sometimes contain 5-6 ringmembers, and the bicyclic heteroaryls sometimes contain 8-10 ringmembers.

Aryl and heteroaryl moieties may be substituted with a variety ofsubstituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl,C5-C1-2 aryl, C1-C8 acyl, and heteroforms of these, each of which canitself be further substituted; other substituents for aryl andheteroaryl moieties include halo, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R,NRCONR₂, NRCOOR, NRCOR, CN, COOR, CONR₂, OOCR, COR, and NO₂, whereineach R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl,and each R is optionally substituted as described above for alkylgroups. The substituent groups on an aryl or heteroaryl group may befurther substituted with the groups described herein as suitable foreach type of such substituents or for each component of the substituent.Thus, for example, an arylalkyl substituent may be substituted on thearyl portion with substituents typical for aryl groups, and it may befurther substituted on the alkyl portion with substituents as typical orsuitable for alkyl groups.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aromatic andheteroaromatic ring systems which are bonded to their attachment pointthrough a linking group such as an alkylene, including substituted orunsubstituted, saturated or unsaturated, cyclic or acyclic linkers. Alinker often is C1-C8 alkyl or a hetero form thereof. These linkers alsomay include a carbonyl group, thus making them able to providesubstituents as an acyl or heteroacyl moiety. An aryl or heteroaryl ringin an arylalkyl or heteroarylalkyl group may be substituted with thesame substituents described above for aryl groups. An arylalkyl groupsometimes includes a phenyl ring optionally substituted with the groupsdefined above for aryl groups and a C1-C4 alkylene that is unsubstitutedor is substituted with one or two C1-C4 alkyl groups or heteroalkylgroups, where the alkyl or heteroalkyl groups can optionally cyclize toform a ring such as cyclopropane, dioxolane, or oxacyclopentane.Similarly, a heteroarylalkyl group often includes a C5-C6 monocyclicheteroaryl group optionally substituted with one or more of the groupsdescribed above as substituents typical on aryl groups and a C1-C4alkylene that is unsubstituted. A heteroarylalkyl group sometimes issubstituted with one or two C1-C4 alkyl groups or heteroalkyl groups, orincludes an optionally substituted phenyl ring or C5-C6 monocyclicheteroaryl and a C1-C4 heteroalkylene that is unsubstituted or issubstituted with one or two C1-C4 alkyl or heteroalkyl groups, where thealkyl or heteroalkyl groups can optionally cyclize to form a ring suchas cyclopropane, dioxolane, or oxacyclopentane.

Where an arylalkyl or heteroarylalkyl group is described as optionallysubstituted, the substituents may be on the alkyl or heteroalkyl portionor on the aryl or heteroaryl portion of the group. The substituentsoptionally present on the alkyl or heteroalkyl portion sometimes are thesame as those described above for alkyl groups, and the substituentsoptionally present on the aryl or heteroaryl portion often are the sameas those described above for aryl groups generally.

“Arylalkyl” groups as used herein are hydrocarbyl groups if they areunsubstituted, and are described by the total number of carbon atoms inthe ring and alkylene or similar linker. Thus a benzyl group is aC7-arylalkyl group, and phenylethyl is a C8-arylalkyl.

“Heteroarylalkyl” as described above refers to a moiety comprising anaryl group that is attached through a linking group, and differs from“arylalkyl” in that at least one ring atom of the aryl moiety or oneatom in the linking group is a heteroatom selected from N, O and S. Theheteroarylalkyl groups are described herein according to the totalnumber of atoms in the ring and linker combined, and they include arylgroups linked through a heteroalkyl linker; heteroaryl groups linkedthrough a hydrocarbyl linker such as an alkylene; and heteroaryl groupslinked through a heteroalkyl linker. Thus, for example,C7-heteroarylalkyl includes pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.

“Alkylene” as used herein refers to a divalent hydrocarbyl group.Because an alkylene is divalent, it can link two other groups together.An alkylene often is referred to as —(CH₂)_(n)— where n can be 1-20,1-10, 1-8, or 1-4, though where specified, an alkylene can also besubstituted by other groups, and can be of other lengths, and the openvalences need not be at opposite ends of a chain. Thus —CH(Me)— and—C(Me)₂— may also be referred to as alkylenes, as can a cyclic groupsuch as cyclopropan-1,1-diyl. Where an alkylene group is substituted,the substituents include those typically present on alkyl groups asdescribed herein.

A suitable linker can be utilized to construct a phospholipid analog(e.g., X²), and multiple linkers are known. Non-limiting examples oflinkers include —(Y)_(y)—, —(Y)_(y)—C(O)N—(Z)_(z)—,—(CH₂)_(y)—C(O)N—(CH₂)_(z)—, —(Y)_(y)—NC(O)—(Z)_(z)—,—(CH₂)_(y)—NC(O)—(CH₂)_(z)—, where each y (subscript) and z (subscript)independently is 0 to 20 and each Y and Z independently is C1-C10 alkyl,substituted C1-C10 alkyl, C1-C10 alkoxy, substituted C1-C10 alkoxy,C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C10 aryl, substitutedC5-C10 aryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic, C1-C6alkanoyl, Het, Het C1-C6 alkyl, or C1-C6 alkoxycarbonyl, wherein thesubstituents on the alkyl, cycloalkyl, alkanoyl, alkcoxycarbonyl, Het,aryl or heterocyclic groups are hydroxyl, C1-C10 alkyl, hydroxyl C1-C10alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C16 alkoxyC16 alkenyl, amino, cyano, halogen or aryl. In certain embodiments, alinker sometimes is a —C(Y′)(Z′)—C(Y″)(Z″)— linker, where each Y′, Y″,Z′ and Z″ independently is hydrogen C1-C10 alkyl, substituted C1-C10alkyl, C1-C10 alkoxy, substituted C1-C10 alkoxy, C3-C9 cycloalkyl,substituted C3-C9 cycloalkyl, C5-C10 aryl, substituted C5-C10 aryl,C5-C9 heterocyclic, substituted C5-C9 heterocyclic, C1-C6 alkanoyl, Het,Het C1-C6 alkyl, or C1-C6 alkoxycarbonyl, wherein the substituents onthe alkyl, cycloalkyl, alkanoyl, alkcoxycarbonyl, Het, aryl orheterocyclic groups are hydroxyl, C1-C10 alkyl, hydroxyl C1-C10alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocyclic, C16 alkoxyC16 alkenyl, amino, cyano, halogen or aryl. Certain non-limitingexamples of linkers that can be utilized include the following:

In some embodiments, a linker is selected that results in a suitableplasma stability. A suitable stability sometimes is about 60% or more(e.g., about 65%, 70%, 75%, 80%, 85%, 90%) of the conjugate analogpresent after contact with human plasma for about 300 minutes.

In general, any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkylgroup, or any heteroform of one of these groups, that is contained in asubstituent may itself optionally be substituted by additionalsubstituents. The nature of these substituents is similar to thoserecited with regard to the primary substituents themselves if thesubstituents are not otherwise described. Thus, where an embodiment of,for example, R¹ is alkyl, this alkyl may optionally be substituted bythe remaining substituents listed as embodiments for R¹ where this makeschemical sense, and where this does not undermine the size limitprovided for the alkyl per se; e.g., alkyl substituted by alkyl or byalkenyl would simply extend the upper limit of carbon atoms for theseembodiments, and is not included. However, alkyl substituted by aryl,amino, alkoxy, ═O, and the like would be included within the scope ofthe invention, and the atoms of these substituent groups are not countedin the number used to describe the alkyl, alkenyl, etc. group that isbeing described. Where no number of substituents is specified, each suchalkyl, alkenyl, alkynyl, acyl, or aryl group may be substituted with anumber of substituents according to its available valences; inparticular, any of these groups may be substituted with fluorine atomsat any or all of its available valences, for example.

“Heteroform” as used herein refers to a derivative of a group such as analkyl, aryl, or acyl, wherein at least one carbon atom of the designatedcarbocyclic group has been replaced by a heteroatom selected from N, Oand S. Thus the heteroforms of alkyl, alkenyl, alkynyl, acyl, aryl, andarylalkyl are heteroalkyl, heteroalkenyl, heteroalkynyl, heteroacyl,heteroaryl, and heteroarylalkyl, respectively. It is understood that nomore than two N, O or S atoms are ordinarily connected sequentially,except where an oxo group is attached to N or S to form a nitro orsulfonyl group. A heteroform moiety sometimes is referred to as “Het”herein.

“Halo” or “halogen,” as used herein includes fluoro, chloro, bromo andiodo. Fluoro and chloro are often preferred. “Amino” as used hereinrefers to NH₂, but where an amino is described as “substituted” or“optionally substituted”, the term includes NR′R″ wherein each R′ and R″is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, orarylalkyl group or a heteroform of one of these groups, and each of thealkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl groups or heteroformsof one of these groups is optionally substituted with the substituentsdescribed herein as suitable for the corresponding group. The term alsoincludes forms wherein R′ and R″ are linked together to form a 3-8membered ring which may be saturated, unsaturated or aromatic and whichcontains 1-3 heteroatoms independently selected from N, O and S as ringmembers, and which is optionally substituted with the substituentsdescribed as suitable for alkyl groups or, if NR′R″ is an aromaticgroup, it is optionally substituted with the substituents described astypical for heteroaryl groups.

As used herein, the term “carbocycle” refers to a cyclic compoundcontaining only carbon atoms in the ring, whereas a “heterocycle” refersto a cyclic compound comprising a heteroatom. The carbocyclic andheterocyclic structures encompass compounds having monocyclic, bicyclicor multiple ring systems. As used herein, the term “heteroatom” refersto any atom that is not carbon or hydrogen, such as nitrogen, oxygen orsulfur. Illustrative examples of heterocycles include but are notlimited to tetrahydrofuran, 1,3 dioxolane, 2,3 dihydrofuran, pyran,tetrahydropyran, benzofuran, isobenzofuran, 1,3 dihydro isobenzofuran,isoxazole, 4,5 dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin 2one, pyrrole, pyridine, pyrimidine, octahydro pyrrolo[3,4b]pyridine,piperazine, pyrazine, morpholine, thiomorpholine, imidazole,imidazolidine 2,4 dione, 1,3 dihydrobenzimidazol 2 one, indole,thiazole, benzothiazole, thiadiazole, thiophene, tetrahydro thiophene1,1 dioxide, diazepine, triazole, guanidine, diazabicyclo[2.2.1]heptane,2,5 diazabicyclo[2.2.1]heptane, 2,3,4,4a,9,9a hexahydro 1H betacarboline, oxirane, oxetane, tetrahydropyran, dioxane, lactones,aziridine, azetidine, piperidine, lactams, and may also encompassheteroaryls. Other illustrative examples of heteroaryls include but arenot limited to furan, pyrrole, pyridine, pyrimidine, imidazole,benzimidazole and triazole.

In some cases, compounds described herein contain one or more chiralcenters. The technology includes each of the isolated stereoisomericforms as well as mixtures of stereoisomers in varying degrees of chiralpurity, including racemic mixtures. It also encompasses the variousdiastereomers and tautomers that can be formed. A compound describedherein also may exist in one or more tautomeric forms. For example, whenR is —OH, a compound described herein may exist in one or moretautomeric forms. A compound described herein can exist as a particularsalt. Non-limiting examples of pharmaceutically acceptable salts aredescribed herein.

The term “optionally substituted” as used herein indicates that theparticular group or groups being described may have no non-hydrogensubstituents, or the group or groups may have one or more non-hydrogensubstituents. If not otherwise specified, the total number of suchsubstituents that may be present is equal to the number of H atomspresent on the unsubstituted form of the group being described. Where anoptional substituent is attached via a double bond, such as a carbonyloxygen (═O), the group takes up two available valences, so the totalnumber of substituents that may be included is reduced according to thenumber of available valences.

Phospholipid Analog Pharmacophores

Provided in certain embodiments are compositions comprising a compoundaccording to Formula E:

P¹—X²—X³—R³  Formula E

or a pharmaceutically acceptable salt or hydrate thereof, where X², X³and R³ are as described above, and P¹ is a pharmacophore.

In some embodiments, provided also are compositions comprising acompound according to Formula F or Formula G:

or a pharmaceutically acceptable salt or hydrate thereof, where X², X³,R³, R^(d) and R^(e) are as described above, and P¹ is a pharmacophore.

With regard to compounds having a structure according to Formula E, F orG, R^(d) and R^(e) independently are a linear and saturated C6-C30 alkylin certain embodiments. In some embodiments R^(d) and R^(e)independently are a C10 to C16 alkyl. In some embodiments R^(d) andR^(e) independently are a C10 to C15, C10 to C14, C10 to C13, C10 to C12or C10 to C11 alkyl. Sometimes R^(d) and R^(e) independently are a C11to C16 alkyl, C11 to C15, C11 to C14, C11 to C13 or C11 to C12 alkyl.Sometimes R^(d) and R^(e) independently are a C12 to C16, C12 to C15,C12 to C14 or C12 to C13 alkyl. Sometimes R^(d) and R^(e) independentlyare a C13 to C16, C13 to C15, C13 to C14, C14 to C16, C14 to C15 or C15to C16 alkyl. Sometimes R^(d) and R^(e) independently are a C10 (e.g., adecyle group), C11 (e.g., an undecyle group), C12 (e.g., a dodecylegroup), C13 (e.g., a tridecyle group), C14 (e.g., a tetradecyle group),C15 (e.g., a pentadecyle group) or C16 alkly (e.g., a hexadecyle group).Sometimes R^(d) and R^(e) independently are a C1-4 alkyl. Each R^(d) andR^(e) often is a linear alkyl group, and each R^(d) and R^(e) often is asaturated alkyl group. In some embodiments R^(d) and R^(e) are the sameor different. In some embodiments, R^(d) and R^(e) independently include1, 2, 3, 4 or 5 double bonds (e.g., unsaturations). In certainembodiments, R^(d) and R^(e) are not substituted by an epoxy moiety, andsometimes R^(d) and R^(e) are not substituted by a hydroxyl moiety. Inspecific embodiments, R^(d) and R^(e) are not substituted by an epoxymoiety or a hydroxyl moiety. In various embodiments, R^(d) and R^(e)include no double bond (e.g., no unsaturation).

With regard to compounds having a structure according to Formula E, F orG, a pharmacophore P¹ can by any molecule that exhibits animmunostimulatory activity. In certain embodiments, a pharmacophore P¹has a structure according to Formula H:

or a pharmaceutically acceptable salt thereof, where a phospholipid, ora phospholipid-like, structure is linked to the pharmacophore at anysuitable linkage point, and where:

-   -   R¹⁰, R²⁰, and R³⁰ in Formula H are each independently hydrogen;        cyclic alkyl of three, four, or five carbon atoms; straight        chain or branched chain alkyl containing one to about ten carbon        atoms and substituted straight chain or branched chain alkyl        containing one to about ten carbon atoms, wherein the        substituent is selected from the group consisting of cycloalkyl        containing three to about six carbon atoms and cycloalkyl        containing three to about six carbon atoms substituted by        straight chain or branched chain alkyl containing one to about        four carbon atoms; fluoro- or chloroalkyl containing from one to        about ten carbon atoms and one or more fluorine or chlorine        atoms; straight chain or branched chain alkenyl containing two        to about ten carbon atoms and substituted straight chain or        branched chain alkenyl containing two to about ten carbon atoms,        wherein the substituent is selected from the group consisting of        cycloalkyl containing three to about six carbon atoms and        cycloalkyl containing three to about six carbon atoms        substituted by straight chain or branched chain alkyl containing        one to about four carbon atoms; hydroxyalkyl of one to about six        carbon atoms; alkoxyalkyl wherein the alkoxy moiety contains one        to about four carbon atoms and the alkyl moiety contains one to        about six carbon atoms; acyloxyalkyl wherein the acyloxy moiety        is alkanoyloxy of two to about four carbon atoms or benzoyloxy,        and the alkyl moiety contains one to about six carbon atoms,        with the proviso that any such alkyl, substituted alkyl,        alkenyl, substituted alkenyl, hydroxyalkyl, alkoxyalkyl, or        acyloxyalkyl group does not have a fully carbon substituted        carbon atom bonded directly to the nitrogen atom; benzyl;        (phenyl)ethyl; and phenyl; said benzyl, (phenyl)ethyl or phenyl        substituent being optionally substituted on the benzene ring by        one or two moieties independently selected from the group        consisting of alkyl of one to about four carbon atoms, alkoxy of        one to about four carbon atoms, and halogen, with the proviso        that when said benzene ring is substituted by two of said        moieties, then the moieties together contain no more than six        carbon atoms; —CHR_(x)R_(y) wherein R_(y) is hydrogen or a        carbon-carbon bond, with the proviso that when R_(y) is hydrogen        R_(x) is alkoxy of one to about four carbon atoms, hydroxyalkoxy        of one to about four carbon atoms, 1-alkynyl of two to about ten        carbon atoms, tetrahydropyranyl, alkoxyalkyl wherein the alkoxy        moiety contains one to about four carbon atoms and the alkyl        moiety contains one to about four carbon atoms, 2-, 3-, or        4-pyridyl, and with the further proviso that when R_(y) is a        carbon-carbon bond R_(y) and R_(x) together form a        tetrahydrofuranyl group optionally substituted with one or more        substituents independently selected from the group consisting of        hydroxy or hydroxyalkyl of one to about four carbon atoms;        straight chain or branched chain alkyl containing one to about        eight carbon atoms, straight chain or branched chain        hydroxyalkyl containing one to about six carbon atoms,        morpholinomethyl, benzyl, (phenyl)ethyl and phenyl, the benzyl,        (phenyl)ethyl or phenyl substituent being optionally substituted        on the benzene ring by a moiety selected from the group        consisting of methyl, methoxy, or halogen; or    -   —C(R_(S))(R_(T))(X) wherein R_(S) and R_(T) are independently        selected from the group consisting of hydrogen, alkyl of one to        about four carbon atoms, phenyl, and substituted phenyl wherein        the substituent is selected from the group consisting of alkyl        of one to about four carbon atoms, alkoxy of one to about four        carbon atoms, and halogen; and    -   X is alkoxy containing one to about four carbon atoms,        alkoxyalkyl wherein the alkoxy moiety contains one to about four        carbon atoms and the alkyl moiety contains one to about four        carbon atoms, haloalkyl of one to about four carbon atoms,        alkylamido wherein the alkyl group contains one to about four        carbon atoms, amino, substituted amino wherein the substituent        is alkyl or hydroxyalkyl of one to about four carbon atoms,        azido, alkylthio of one to about four carbon atoms, or        morpholinoalkyl wherein the alkyl moiety contains one to about        four carbon atoms;    -   R⁴⁰ in Formula H is hydrogen, C₁₋₈ alkyl, C₁₋₈ alkoxy, or halo;    -   nn in Formula H is 1, 2, 3, or 4;    -   R^(aa) and R^(bb) in Formula H are each independently hydrogen,        (C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl, adamantyl,        adamantyl(C₁-C₆)alkyl, amino(C₁-C₆)alkyl, aminosulfonyl,        (C₁-C₆)alkanoyl, aryl, or benzyl; or R^(aa) and R^(bb) together        with the nitrogen to which they are attached form a pyrrolidino,        piperidino, or morpholino group; and    -   the dashed lines in the five membered ring of Formula H denote        an optional bond that connects a nitrogen of the five membered        ring to the carbon that is between the two nitrogens of the five        membered ring, and when the bond is present, either R¹⁰ or R³⁰        is absent.

In some embodiments, one of R^(aa) or R^(bb) in Formula H independentlyis —X²—X³—R³, or has a structure according to Formula C or Formula D,and the other R^(aa) or R^(bb) is hydrogen, C1-C6 alkyl or C1-C6 alkoxy.

In certain embodiments, a pharmacophore P¹ has a structure according toFormula I:

or a pharmaceutically acceptable salt thereof, where a phospholipid, ora phospholipid-like, structure is linked to the pharmacophore at anysuitable linkage point, and where R^(aa) and R^(bb) are as definedabove. In some embodiments, one of R^(aa) or R^(bb) in Formula I is—X²—X³—R³, or has a structure according to Formula C or Formula D, andthe other R^(aa) or R^(bb) is hydrogen, C1-C6 alkyl or C1-C6 alkoxy.

Also with regard to compounds having a structure according to Formula E,F or G, a pharmacophore P¹, in certain embodiments, has a structureaccording to Formula J or Formula K:

or a pharmaceutically acceptable salt thereof, where a phospholipid, ora phospholipid-like, structure is linked to the pharmacophore at anysuitable linkage point, where R⁴⁰, nn, R^(aa) and R^(bb) are as definedabove, and where R^(cc) is hydrogen, C1-C6 alkyl or C1-C6 alkoxy. Insome embodiments, one of R^(aa) or R^(bb) in Formula J or Formula K is—X²—X³—R³, or has a structure according to Formula C or Formula D, andthe other R^(aa) or R^(bb) is hydrogen, C1-C6 alkyl or C1-C6 alkoxy. Inthe latter embodiments R^(cc) sometimes is hydrogen. In certainembodiments, R^(cc) n Formula J or Formula K is —X²—X³—R³, or has astructure according to Formula C or Formula D. In the latterembodiments, R^(aa) or R^(bb) independently are hydrogen, C1-C6 alkyl orC1-C6 alkoxy.

Pharmaceutical Compositions and Formulations

A compound described herein can be prepared as a pharmaceuticallyacceptable salt. As used herein, the term “pharmaceutically acceptablesalt” refers to a derivative of the disclosed compounds where the parentcompound is modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Pharmaceutically acceptable salts include conventional non-toxicsalts or quaternary ammonium salts of the parent compound formed, forexample, from non-toxic inorganic or organic acids. For example,conventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. In other examples, conventional non-toxic salts includethose derived from bases, such as potassium hydroxide, sodium hydroxide,ammonium hydroxide, caffeine, various amines, and the like.Pharmaceutically acceptable salts can be synthesized from the parentcompound, which contains a basic or acidic moiety, by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., p. 1418 (1985), thedisclosure of which is hereby incorporated by reference.

The term “pharmaceutically acceptable” as used herein refers tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. Stable compounds are contemplated hereinfor use in treatment methods described.

A compound described herein can be formulated in combination with one ormore other agents. The one or more other agents can include, withoutlimitation, another compound described herein, an anti-cellproliferative agent (e.g., chemotherapeutic), an anti-inflammatoryagent, and an antigen.

A compound described herein can be formulated as a pharmaceuticalcomposition and administered to a mammalian host, such as a humanpatient or nonhuman animal, in a variety of forms adapted to the chosenroute of administration. Non-limiting examples of routes ofadministration include oral, parenteral, intravenous, intramuscular,topical, instillation (e.g., bladder instillation), subcutaneous,intradermal routes. In certain embodiments, a composition is locallyadministered, e.g., intravesicularly. A composition sometimes includes adiluent and sometimes an adjuvant, carrier (e.g., assimilable,editable), buffer, preservative and the like. A compound can beadministered also in a liposomal composition or as a microemulsion, incertain embodiments. Various sustained release systems for drugs havealso been devised, and can be applied to a compound described herein.See, for example, U.S. Pat. No. 5,624,677, the methods of which areincorporated herein by reference.

For administration to the bladder of a subject, in some embodiments, aconcentration of about 10 nM to about 1000 nM, or about 100 nM to about10,000 nM, of a compound described herein may be delivered. In certainembodiments, a composition described herein is administered inconjunction with locally applied ultrasound, electromagnetic radiationor electroporation or other electrically based drug delivery technique,local chemical abrasion, or local physical abrasion. In someembodiments, a composition described herein includes, or is administeredwith, a surfactant (e.g., a locally applied) to enhance permeability ofa compound described herein across the bladder mucosa. In certainembodiments, a composition herein provides enhanced endosomal uptake,which can result from particle size, receptor multimerization orsustained release, for example.

Compounds described herein may be enclosed in hard or soft shell gelatincapsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, an active compound may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations sometimes contain at least 0.1% ofactive compound. The percentage of the compositions and preparations maybe varied and sometimes are about 2% to about 60% of the weight of agiven unit dosage form. The amount of active compound in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

Tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

An active compound may be administered by infusion or injection.Solutions of an active compound or a pharmaceutically acceptable saltthereof can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations sometimescontain a preservative to prevent the growth of microorganisms.

A pharmaceutical dosage form can include a sterile aqueous solution ordispersion or sterile powder comprising an active ingredient, which areadapted for the extemporaneous preparation of sterile solutions ordispersions, and optionally encapsulated in liposomes. The ultimatedosage form sometimes is a sterile fluid and stable under the conditionsof manufacture and storage. A liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. An isotonic agent, for example, a sugar, buffer or sodiumchloride is included in some embodiments. Prolonged absorption of aninjectable composition can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin. Sterile solutions often are prepared byincorporating an active compound in a required amount in an appropriatesolvent, sometimes with one or more of the other ingredients enumeratedabove, followed by filter sterilization. In the case of sterile powdersfor the preparation of sterile injectable solutions, preparation methodssometimes utilized are vacuum drying and the freeze drying techniques,which yield a powder of an active ingredient in addition to anyadditional desired ingredient present in the previously sterile-filteredsolutions.

For topical administration, a compound herein may be applied in pureform, e.g., when in liquid form. However, it is generally desirable toadminister a compound as a composition or formulation, in combinationwith an acceptable carrier, which may be a solid or a liquid. Usefulsolid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, or phospholipids in propylenglycol/ethylenglycol, in which thepresent compounds can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

The ability of a compound herein to act as a TLR agonist or TLRantagonist may be determined using pharmacological models which areknown, including the procedures disclosed by Lee et al., PNAS, 100:6646(2003).

Useful dosages of compounds can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art. In some embodiments, the concentration of acompound described herein in a liquid composition is about 0.1-25 wt-%,and sometimes about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder sometimes is about 0.1-5wt-%, and sometimes about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment varies not only with a particular saltselected but also with the route of administration, the nature of thecondition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. In general a suitable dose sometimes is in the range of fromabout 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg ofbody weight per day, such as 3 to about 50 mg per kilogram body weightof the recipient per day, and often is in the range of 6 to 90mg/kg/day, or about 15 to 60 mg/kg/day. A suitable dose, in general,sometimes is in the range of from about 1 to 150 mg/kg body weight ofthe recipient per day, e.g. from about 10 to about 130 mg/kg, from about40 to about 120 mg/kg, from about 50 to about 100 mg/kg, from about 60to 90 mg/kg, from about 65 to 85 mg/kg, or, for example, about 80mg/kg/day. A compound may be conveniently administered in unit dosageform, and for example, contain 5 to 1000 mg, or 10 to 750 mg, or 50 to500 mg of active ingredient per unit dosage form. An active ingredientcan be administered to achieve peak plasma concentrations of an activecompound of from about 0.01 to about 100 pM, about 0.5 to about 75 pM,about 1 to 50 pM, or about 2 to about 30 pM. Such concentrations may beachieved, for example, by the intravenous injection of a 0.05 to 5%solution of an active ingredient, optionally in saline, or orallyadministered as a bolus containing about 1-100 mg of an activeingredient. Desirable blood levels may be maintained by continuousinfusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusionscontaining about 0.4-15 mg/kg of the active ingredient(s). A desireddose may conveniently be presented in a single dose or as divided dosesadministered at appropriate intervals, for example, as two, three, fouror more sub-doses per day. A sub-dose itself may be further divided,e.g., into a number of discrete loosely spaced administrations; such asmultiple inhalations from an insufflator or by application of aplurality of drops into the eye.

Treatments

Compositions provided may be useful for the treatment or prevention ofcertain conditions in a subject. Such conditions include, for example,proliferative conditions such as cancers, microbial infections, heartconditions and obesity conditions; inflammation conditions andautoimmune conditions in certain embodiments.

The terms “treat” and “treating” as used herein refer to (i) preventinga pathologic condition from occurring (e.g. prophylaxis); (ii)inhibiting the pathologic condition or arresting its development; (iii)relieving the pathologic condition; and/or (iv) ameliorating,alleviating, lessening, and removing symptoms of a disease or condition.A candidate molecule or compound described herein may be in atherapeutically effective amount in a formulation or medicament, whichis an amount that can lead to a biological effect (e.g., inhibitinginflammation), or lead to ameliorating, alleviating, lessening,relieving, diminishing or removing symptoms of a disease or condition,for example. The terms also can refer to reducing or stopping a cellproliferation rate (e.g., slowing or halting tumor growth) or reducingthe number of proliferating cancer cells (e.g., removing part or all ofa tumor). A molecule described herein can be administered to a subjectin need thereof to potentially treat a melanoma. In such treatments, theterms “treating,” “treatment” and “therapeutic effect” can refer toreducing or stopping a cell proliferation rate (e.g., slowing or haltingtumor growth), reducing the number of proliferating cancer cells (e.g.,ablating part or all of a tumor) and alleviating, completely or in part,a melanoma condition.

A drug, which can be a prophylactic or therapeutic agent, can beadministered to any appropriate subject having a melanoma as describedherein. Non-limiting examples of a subject include mammal, human, ape,monkey, ungulate (e.g., equine, bovine, caprine, ovine, porcine,buffalo, camel and the like), canine, feline, rodent (e.g., murine,mouse, rat) and the like. A subject may be male or female, and a drugcan be administered to a subject in a particular age group, including,for example, juvenile, pediatric, adolescent, adult and the like.

The term “therapeutically effective amount” as used herein refers to anamount of a compound provided herein, or an amount of a combination ofcompounds provided herein, to treat or prevent a disease or disorder, orto treat a symptom of the disease or disorder, in a subject. As usedherein, the terms “subject” and “patient” generally refers to anindividual who will receive or who has received treatment (e.g.,administration of a compound described herein) according to a methoddescribed herein.

A proliferative condition sometimes is a cancer. Cancers and relateddisorders sometimes are of an epithelial cell origin. In someembodiments, a proliferative condition is associated with blood, such asleukemia. Non-limiting examples of leukemias and other blood conditionsinclude acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic,and erythroleukemia leukemias) and myelodysplastic syndrome; chronicleukemias, such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia; andpolycythemia vera.

In certain embodiments, a proliferative condition presents as alymphoma. Non-limiting examples of lymphomas include Hodgkin's diseaseand non-Hodgkin's disease. A proliferative condition sometimes is amultiple myeloma, non-limiting examples of which include smolderingmultiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasmacell leukemia, solitary plasmacytoma and extramedullary plasmacytoma. Aproliferative condition in some embodiments presents as Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; or heavy chain disease.

A proliferative condition in some embodiments presents as a sarcoma(e.g., in bone or connective tissue). Non-limiting examples of sarcomasinclude bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma.

In some embodiments, a proliferative condition presents as a conditionof the brain (e.g., brain tumor). Non-limiting examples of proliferativeconditions of the brain include glioma, astrocytoma, brain stem glioma,ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma.

A proliferative condition in some embodiments is a breast cancer.Non-limiting breast cancers include ductal carcinoma, adenocarcinoma,lobular (small cell) carcinoma, intraductal carcinoma, medullary breastcancer, mucinous breast cancer, tubular breast cancer, papillary breastcancer, Paget's disease, and inflammatory breast cancer. In certainembodiments, a proliferative condition presents as an adrenal cancer.Non-limiting examples of adrenal cancer include pheochromocytom andadrenocortical carcinoma. A proliferative condition sometimes presentsas a thyroid cancer, including, but not limited to papillary orfollicular thyroid cancer, medullary thyroid cancer and anaplasticthyroid cancer.

In certain embodiments, a proliferative condition presents as apancreatic cancer, including, but not limited to, insulinoma,gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, andcarcinoid or islet cell tumor. A proliferative condition in someembodiments presents as a pituitary cancer, non-limiting examples ofwhich include Cushing's disease, prolactin-secreting tumor, acromegaly,and diabetes insipius. In some embodiments, a proliferative conditionpresents as an eye cancer, including but not limited to, ocular melanomasuch as iris melanoma, choroidal melanoma, and cilliary body melanoma,and retinoblastoma.

A proliferative condition in certain embodiments presents as a vaginalcancer or vulvar cancer, which can include without limitation squamouscell carcinoma, adenocarcinoma, melanoma, squamous cell carcinoma,melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget'sdisease. In some embodiments, a proliferative condition presents as acervical cancers, which can include, but is not limited to, squamouscell carcinoma and adenocarcinoma. Uterine cancers also are a form ofcertain proliferative conditions, including, but not limited to,endometrial carcinoma and uterine sarcoma. A proliferative conditionsometimes is an ovarian cancer, non-limiting examples of which includeovarian epithelial carcinoma, borderline tumor, germ cell tumor, andstromal tumor.

In some embodiments, a proliferative condition is an esophageal cancer,non-limiting examples of which include squamous cancer, adenocarcinoma,adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamouscarcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oatcell (small cell) carcinoma. A proliferative condition sometimespresents as a stomach cancer, including, but not limited to,adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading,diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma. A proliferative condition sometimes presents as coloncancer or a rectal cancers. In some embodiments, a proliferativecondition is a liver cancer, non-limiting examples of which includehepatocellular carcinoma and hepatoblastoma. A proliferative conditionin certain embodiments presents as a gallbladder cancer, including, butnot limited to, adenocarcinoma. In certain embodiments, a proliferativecondition presents as bile duct cancer, such as cholangiocarcinomas(e.g., papillary, nodular, and diffuse) for example.

A proliferative condition in some embodiments is a lung cancer.Non-limiting examples of lung cancers include non-small cell lungcancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer. In certain embodiments,a proliferative condition presents as a testicular cancer, such as agerminal tumor, seminoma, anaplastic, classic (typical), spermatocytic,nonseminoma, embryonal carcinoma, teratoma carcinoma or choriocarcinoma(yolk-sac tumor). A proliferative condition in some embodiments is aprostate cancer, including, but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma. In certain embodiments, a proliferative condition is apenal cancer.

A proliferative condition sometimes is an oral cancer, non-limitingexamples of which include squamous cell carcinoma; basal cancers;salivary gland cancers such as but not limited to adenocarcinoma,mucoepidermoid carcinoma, and adenoidcystic carcinoma. In someembodiments, a proliferative condition is a pharynx cancers, including,but not limited to squamous cell cancer and verrucous. A proliferativecondition sometimes presents as a skin cancer, non-limiting examples ofwhich include basal cell carcinoma, squamous cell carcinoma, melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, and acral lentiginous melanoma.

In some embodiments, a proliferative condition is a kidney cancer suchas a renal cell carcinoma, adenocarcinoma, hypemephroma, fibrosarcoma,transitional cell cancer (renal pelvis and/or uterer), and Wilms' tumor.In certain embodiments, a proliferative condition is a bladder cancer,non-limiting examples of which include superficial bladder cancer,transitional cell carcinoma, squamous cell cancer, adenocarcinoma andcarcinosarcoma.

In certain embodiments, a proliferative condition is a cancer selectedfrom myxosarcoma, osteogenic sarcoma, endotheliosarcoma,lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma,epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma andpapillary adenocarcinomas; carcinoma, including that of the bladder,breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix,thyroid and skin (e.g., squamous cell carcinoma); hematopoietic tumorsof lymphoid lineage, including leukemia, acute lymphocytic leukemia,acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma,Burkitt's lymphoma; hematopoictic tumors of myeloid lineage, includingacute and chronic myelogenous leukemias and promyclocytic leukemia;tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis can be addressed by compositions describedherein. Such cancers may include but not be limited to follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, and precancerous lesions such asfamilial adenomatous polyposis, and myelodysplastic syndromes. Inspecific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders, may betreated or prevented in the skin, lung, colon, breast, prostate,bladder, kidney, pancreas, ovary, or uterus.

Cell proliferative conditions also include viral diseases, including forexample, acquired immunodeficiency syndrome, adenoviridae infections,alphavirus Infections, arbovirus Infections, Borna disease, bunyaviridaeInfections, caliciviridae Infections, chickenpox, CoronaviridaeInfections, coxsackievirus Infections, cytomegalovirus Infections,dengue, DNA Virus Infections, eethyma, contagious, encephalitis,arbovirus, Epstein-Barr virus infections, erythema infectiosum,hantavirus infections, hemorrhagic fevers, viral, hepatitis, viral,human, herpes simplex, herpes zoster, herpes zoster oticus,herpesviridae infections, infectious mononucleosis, influenza, e.g., inbirds or humans, Lassa fever, measles, Molluscum contagiosum, mumps,oaramyxoviridae Infections, phlebotomus fever, polyomavirus infections,rabies, respiratory syncytial virus Infections, Rift Valley fever, RNAVirus Infections, rubella, slow virus diseases, smallpox, subacutesclerosing panencephalitis, tumor virus infections, warts, West Nilefever, virus diseases and Yellow Fever. For example, Large T antigen ofthe SV40 transforming virus acts on UBF, activates it and recruits otherviral proteins to Pol I complex, and thereby stimulates cellproliferation to ensure virus propagation. Cell proliferative conditionsalso include conditions related to angiogenesis (e.g., cancers) andobesity caused by proliferation of adipocytes and other fat cells.

Cell proliferative conditions include microbial infections. Non-limitingexamples of microbes include viruses, bacteria, yeast and fungus.Examples of certain microbes that may be treated by a compositiondescribed are listed herein.

Cell proliferative conditions also include cardiac conditions resultingfrom cardiac stress, such as hypertension, balloon angioplasty, valvulardisease and myocardial infarction. For example, cardiomyocytes aredifferentiated muscle cells in the heart that constitute the bulk of theventricle wall, and vascular smooth muscle cells line blood vessels.Although both are muscle cell types, cardiomyocytes and vascular smoothmuscle cells vary in their mechanisms of contraction, growth anddifferentiation. Cardiomyocytes become terminally differentiated shortlyafter heart formation and thus loose the capacity to divide, whereasvascular smooth muscle cells are continually undergoing modulation fromthe contractile to proliferative phenotype. Under variouspathophysiological stresses such as hypertension, balloon angioplasty,valvular disease and myocardial infarction, for example, the heart andvessels undergo morphologic growth-related alterations that can reducecardiac function and eventually manifest in heart failure. Thus,provided herein are methods for treating cardiac cell proliferativeconditions by administering a compound described herein in an effectiveamount to treat the cardiac condition. A compound may be administeredbefore or after a cardiac stress has occurred or has been detected, andthe compound or nucleic acid may be administered after occurrence ordetection of hypertension, balloon angioplasty, valvular disease ormyocardial infarction, for example. Administration of such a compoundmay decrease proliferation of vascular muscle cells and/or smooth musclecells.

A cell proliferation condition also may pertain to obesity. In someembodiments, a cell proliferative condition is abnormal proliferation ofadipocytes.

A compound described herein can be administered to a subject in needthereof to induce an immune response in the subject. The immune responsemay be generated automatically by the subject against a foreign antigen(e.g., pathogen infection) in certain embodiments. In some embodiments,an antigen is co-administered with a compound described herein, where animmune response is mounted in the subject against the antigen. Anantigen may be specific for a particular cell proliferative condition(e.g., specific cancer antigen) or particular pathogen (e.g., grampositive bacteria wall antigen; S. aureus antigen), in certainembodiments. An immunostimulatory composition may be administered in avaccine or combination vaccine, in some embodiments. Animmunostimulatory composition may be administered as an adjuvantcomposition in certain embodiments, and may be administered inconjunction with an antigen (e.g., sequential administration orco-administration with antigen) in certain embodiments.

A compound described herein can be administered to a subject in needthereof to potentially prevent, inhibit or treat one or moreinflammation disorders. As used hereinafter, the terms “treating,”“treatment” and “therapeutic effect” can refer to reducing, inhibitingor stopping (preventing) an inflammation response (e.g., slowing orhalting antibody production or amount of antibodies to a specificantigen), reducing the amount of inflamed tissue and alleviating,completely or in part, an inflammation condition. Inflammation disordersinclude, without limitation, allergy, asthma, autoimmune disorder,chronic inflammation, chronic prostatitis, glomerulonephritis,hypersensitivities, inflammatory bowel diseases, myopathy (e.g., incombination with systemic sclerosis, dermatomyositis, polymyositis,and/or inclusion body myositis), pelvic inflammatory disease,reperfusion injury, rheumatoid arthritis, transplant rejection,vasculitis, and leukocyte disorders (e.g., Chediak-Higashi syndrome,chronic granulomatous disease). Certain autoimmune disorders also areinflammation disorders (e.g., rheumatoid arthritis). In someembodiments, the inflammation disorder is selected from the groupconsisting of chronic inflammation, chronic prostatitis,glomerulonephritis, a hypersensitivity, myopathy, pelvic inflammatorydisease, reperfusion injury, transplant rejection, vasculitis, andleukocyte disorder. In certain embodiments, an inflammation conditionincludes, but is not limited to, bronchiectasis, bronchiolitis, cysticfibrosis, acute lung injury, acute respiratory distress syndrome (ARDS),atherosclerosis, and septic shock (e.g., septicemia with multiple organfailure). In some embodiments, an inflammation disorder is not acondition selected from the group consisting of allergy, asthma, ARDSand autoimmune disorder. In certain embodiments, an inflammationdisorder is not a condition selected from the group consisting ofgastrointestinal tract inflammation, brain inflammation, skininflammation and joint inflammation. In certain embodiments, theinflammation disorder is a neutrophil-mediated disorder. In someembodiments, an inflammatory condition also is a cell proliferationcondition, such as, for example, inflammation conditions of the skin(e.g., eczema), discoid lupus erythematosus, lichen planus, lichensclerosus, mycosis fungoides, photodermatoses, pityriasis rosea andpsoriasis.

A compound described herein can be administered to a subject in needthereof to potentially treat one or more autoimmune disorders. In suchtreatments, the terms “treating,” “treatment” and “therapeutic effect”can refer to reducing, inhibiting or stopping an autoimmune response(e.g., slowing or halting antibody production or amount of antibodies toa specific antigen), reducing the amount of inflamed tissue andalleviating, completely or in part, an autoimmune condition. Autoimmunedisorders, include, without limitation, autoimmune encephalomyelitis,colitis, automimmune insulin dependent diabetes mellitus (IDDM), andWegener granulomatosis and Takayasu arteritis. Models for testingcompounds for such diseases include, without limitation, (a)(i) C5BL/6induced by myelin oligodendrocyte glycoprotein (MOG) peptide, (ii) SJLmice PLP139-151, or 178-191 EAE, and (iii) adoptive transfer model ofEAE induced by MOG or PLP peptides for autoimmune encephalomyelitis; (b)non-obese diabetes (NOD) mice for autoimmune IDDM; (c) dextran sulfatesodium (DSS)-induced colitis model and trinitrobenzene sulfonic acid(TNBS)-induced colitis model for colitis; and (d) systemic smallvasculitis disorder as a model for Wegener granulomatosis and Takayasuarteritis. A compound described herein may be administered to a subjectto potentially treat one or more of the following disorders: Acutedisseminated encephalomyelitis (ADEM); Addison's disease; alopeciagreata; ankylosing spondylitis; antiphospholipid antibody syndrome(APS); autoimmune hemolytic anemia; autoimmune hepatitis; autoimmuneinner ear disease; bullous pemphigoid; celiac disease; Chagas disease;chronic obstructive pulmonary disease; Crohns disease (one of two typesof idiopathic inflammatory bowel disease “IBD”); dermatomyositis;diabetes mellitus type 1; endometriosis; Goodpasture's syndrome; Graves'disease; Guillain-Barré syndrome (GBS); Hashimoto's disease;hidradenitis suppurativa; idiopathic thrombocytopenic purpura;interstitial cystitis; lupus erythematosus; mixed connective tissuedisease; morphea; multiple sclerosis (MS); myasthenia gravis;narcolepsy; neuromyotonia; pemphigus vulgaris; pernicious anaemia;polymyositis; primary biliary cirrhosis; rheumatoid arthritis;schizophrenia; scleroderma; Sjögren's syndrome; temporal arteritis (alsoknown as “giant cell arteritis”); ulcerative colitis (one of two typesof idiopathic inflammatory bowel disease “IBD”); vasculitis; vitiligo;and Wegener's granulomatosis. In some embodiments, the autoimmunedisorder is not a condition selected from the group consisting of Crohnsdisease (or Crohn's disease), rheumatoid arthritis, lupus and multiplesclerosis.

In some embodiments, a compound described herein is utilized incombination with administration of one or more other therapies thatinclude, but are not limited to, chemotherapies, radiation therapies,hormonal therapies, and/or biological therapies (e.g. immunotherapies).An agent that can be used in combination with a compound describedherein can include, but is not limited to, a proteinaceous molecule,including, but not limited to, peptide, polypeptide, protein, includingpost-translationally modified protein, antibody and the like; smallmolecule (less than 1000 daltons); inorganic or organic compounds;nucleic acid molecule, including, but not limited to, double-stranded orsingle-stranded DNA, or double-stranded or single-stranded RNA, andtriple helix nucleic acid molecules. An agent used in combination with acompound described herein can be derived from any known organism(including, but not limited to, animals, plants, bacteria, fungi, andprotista, or viruses) or from a library of synthetic molecules. An agentthat may be utilized in combination with a compound described hereinincludes a protein kinase inhibitor (e.g., a receptor protein kinaseinhibitor) and an angiogenesis inhibitor.

Immunostimulatory Compositions

Compounds described herein may have immunostimulatory activity, and canenhance the level of an immune response against an antigen. Accordingly,a compound described herein may be useful as an adjuvant that can beadministered in conjunction with an antigen. Accordingly, a compounddescribed herein can be incorporated as part of a vaccine compositionthat contains an antigen in some embodiments, and can be administeredseparately from an antigen in an adjuvant composition in certainembodiments. Vaccine compositions and adjuvant compositions are referredto collectively herein as “immunostimulatory compositions.”

Immunostimulatory Composition Components

A compound described herein can be utilized in an immunostimulatorycomposition in any effective amount. In certain embodiments, a compounddescribed herein can be used in an amount of about 1 micrograms to about100,000 micrograms per dose. A compound described herein also can beused in an amount of about 1 micrograms to about 50,000 micrograms perdose, about 1 micrograms to about 25,000 micrograms per dose, about 1micrograms to about 5,000 micrograms per dose, about 1 micrograms toabout 4,000 micrograms per dose, about 1 micrograms to about 3,000micrograms per dose, about 1 micrograms to about 2,000 micrograms perdose, and about 1 micrograms to about 1,000 micrograms per dose. Acompound described herein also may be used in an amount of about 5micrograms to about 750 micrograms per dose, about 5 micrograms to about500 micrograms per dose, about 5 micrograms to about 200 micrograms perdose, about 5 micrograms to about 100 micrograms per dose, about 15micrograms to about 100 micrograms per dose, and in an amount of about30 micrograms to about 75 micrograms per dose, in some embodiments.

In addition to a compound described herein, an immunostimulatorycomposition can include one or more other components. For example, atriterpenoid can be included in an immunostimulatory composition.Triterpenoids suitable for use in an immunostimulatory composition cancome from many sources (e.g., plant derived or synthetic equivalents),including but not limited to, Quillaja saponaria, tomatine, ginsengextracts, mushrooms, and an alkaloid glycoside structurally similar tosteroidal saponins. Thus, triterpenoids suitable for use in animmunostimulatory composition include saponins, squalene, andlanosterol. The amount of a triterpenoid suitable for use in animmunostimulatory composition depends upon the nature of thetriterpenoid used. However, they are generally used in an amount ofabout 1 micrograms to about 5,000 micrograms per dose. They also can beused in an amount of about 1 micrograms to about 4,000 micrograms perdose, about 1 micrograms to about 3,000 micrograms per dose, about 1micrograms to about 2,000 micrograms per dose, and about 1 micrograms toabout 1,000 micrograms per dose. They also may be used in an amount ofabout 5 micrograms to about 750 micrograms per dose, about 5 microgramsto about 500 micrograms per dose, about 5 micrograms to about 200micrograms per dose, about 5 micrograms to about 100 micrograms perdose, about 15 micrograms to about 100 micrograms per dose, and in anamount of about 30 micrograms to about 75 micrograms per dose.

If a saponin is used, an immunostimulatory composition often contains animmunologically active saponin fraction from the bark of Quillajasaponaria. The saponin may be, for example, Quil A or another purifiedor partially purified saponin preparation, which can be obtainedcommercially. Thus, saponin extracts can be used as mixtures or purifiedindividual components such as QS-7, QS-17, QS-18, and QS-21. In someembodiments the Quil A is at least 85% pure. In other embodiments, theQuil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%pure.

CpG oligodeoxynucleic acids are characterized by the presence of anunmethylated CG dinucleotide in specific base-sequence contexts (CpGmotif), and can confer immunostimulatory properties. Theseimmunostimulatory properties include induction of a Th1-type responsewith prominent release of IFN-, IL-12, and IL-18. CpG ODNs (18-24 bp inlength). A carrier such as QCDC, QCDCR and other combinations canfacilitate uptake of CpG oligodeoxynucleic acids. The amount of CpG foruse in an immunostimulatory composition depends upon the nature of theCpG used and the intended species. However, they often are used in anamount of about 1 micrograms to about 20 mg per dose. They also can beused in an amount of about 1 micrograms to about 10 mg per dose, about 1micrograms to about 5 mg per dose, about 1 micrograms to about 4 mg perdose, about 1 micrograms to about 3 mg per dose, about 1 micrograms toabout 2 mg per dose, and about 1 micrograms to about 1 mg per dose. Theyare can be used in an amount of about 5 micrograms to about 750micrograms per dose, about 5 micrograms to about 500 micrograms perdose, about 5 micrograms to about 200 micrograms per dose, about 5micrograms to about 100 micrograms per dose, 10 micrograms to about 100micrograms per dose, about 15 micrograms to about 100 micrograms perdose, and in an amount of about 30 micrograms to about 75 micrograms perdose.

Sterols also can be used in an immunostimulatory composition, andsterols suitable for use include beta-sitosterol, stigmasterol,ergosterol, ergocalciferol, and cholesterol. These sterols are known inthe art and can be purchased commercially. The amount of sterolssuitable for use in an immunostimulatory composition depends upon thenature of the sterol used. However, they are often used in an amount ofabout 1 micrograms to about 5,000 micrograms per dose. They also can beused in an amount of about 1 micrograms to about 4,000 micrograms perdose, about 1 micrograms to about 3,000 micrograms per dose, about 1micrograms to about 2,000 micrograms per dose, and about 1 micrograms toabout 1,000 micrograms per dose. They also can be used in an amount ofabout 5 micrograms to about 750 micrograms per dose, about 5 microgramsto about 500 micrograms per dose, about 5 micrograms to about 200micrograms per dose, about 5 micrograms to about 100 micrograms perdose, about 15 micrograms to about 100 micrograms per dose, and about 30micrograms to about 75 micrograms per dose.

An immunostimulatory composition can further include one or moreimmunomodulatory agents, non-limiting examples of which includequaternary ammonium compounds (e.g., DDA), and interleukins,interferons, or other cytokines. These materials can be purchasedcommercially. The amount of an immunomodulator suitable for use in animmunostimulatory composition depends upon the nature of theimmunomodulator used and the subject. However, they often are used in anamount of about 1 micrograms to about 5,000 micrograms per dose. Theyalso can be used in an amount of about 1 micrograms to about 4,000micrograms per dose, about 1 micrograms to about 3,000 micrograms perdose, about 1 micrograms to about 2,000 micrograms per dose, and about 1micrograms to about 1,000 micrograms per dose. They also can be used inan amount of about 5 micrograms to about 750 micrograms per dose, about5 micrograms to about 500 micrograms per dose, about 5 micrograms toabout 200 micrograms per dose, about 5 micrograms to about 100micrograms per dose, about 15 micrograms to about 100 micrograms perdose, and in an amount of about 30 micrograms to about 75 micrograms perdose. In a specific example, an immunostimulatory composition containingDDA can be prepared by simply mixing an antigen solution with a freshlyprepared solution of DDA.

An immunostimulatory composition can further include one or morepolymers, non-limiting examples of which include DEAE Dextran,polyethylene glycol, and polyacrylic acid and polymethacrylic acid (eg,CARBOPOL®). The amount of polymer suitable for use in animmunostimulatory composition depends upon the nature of the polymersused. However, they often are used in an amount of about 0.0001% volumeto volume (v/v) to about 75% v/v. In some embodiments, they are used inan amount of about 0.001% v/v to about 50% v/v, of about 0.005% v/v toabout 25% v/v, of about 0.01% v/v to about 10% v/v, of about 0.05% v/vto about 2% v/v, and of about 0.1% v/v to about 0.75% v/v. In certainembodiments, they are used in an amount of about 0.02 v/v to about 0.4%v/v. DEAE-dextran can have a molecular size in the range of 50,000 Da to5,000,000 Da, or it can be in the range of 500,000 Da to 2,000,000 Da.Such material may be purchased commercially or prepared from dextran.

In some embodiments, a polymer utilized is polyacrylic acid (e.g., theCARBOPOL® polymers), which has an average equivalent weight of 76.Polyacrylic acids often are produced from primary polymer particles ofabout 0.2 to 6.0 microns in average diameter. The CARBOPOL® polymersswell in water up to 1000 times their original volume and ten timestheir original diameter to form a gel when exposed to a pH environmentgreater than the pKa of the carboxylate group. At a pH greater than thepKa of carboxylate group, the carboxylate groups ionize resulting inrepulsion between the negative charges, which adds to the swelling ofthe polymer.

An immunostimulatory composition can further include one or more Th2stimulants such as, for example, Bay R1005® and aluminum. The amount ofTh2 stimulants suitable for use in an immunostimulatory compositiondepends upon the nature of the Th2 stimulant used. However, a Th2stimulant often is used in an amount of about 0.01 mg to about 10 mg perdose. In some embodiments, such stimulants are used in an amount ofabout 0.05 mg to about 7.5 mg per dose, of about 0.1 mg to about 5 mgper dose, of about 0.5 mg to about 2.5 mg per dose, and of 1 mg to about2 mg per dose. A specific example is Bay R1005®, a glycolipid with thechemical name“N-(2-deoxy-2-L-leucylamino-.beta.-D-glucopyranosyl)-N-octadecyldodecanam-ideacetate.” It can be synthesized according to the procedure known in theart. It often is stored at 2-8 degrees Celsius in an airtight container.Its chemical or physical properties are that it is slightly hygroscopic,does not form polymorphs, is chemically stable in air and light attemperatures up to 50 degrees Celsius and in aqueous solvents at pH 2-12at ambient temperature. It is an amphiphilic molecule which formsmicelles in aqueous solution.

Antigens

An immunostimulatory composition can contain one or more antigens. Theantigen can be any of a wide variety of substances capable of producinga desired immune response in a subject. Although Quil A alone isvirucidal, Quil A is detoxified with the addition of cholesterol whenforming helical micelles. An immunostimulatory composition can benon-viricidal, and non-hemolytic or membranolytic. Thus, an antigensused with a immunostimulatory composition can be one or more of viruses(inactivated, attenuated, and modified live), bacteria, parasites,nucleotides, polynucleotides, peptides, polypeptides, recombinantproteins, synthetic peptides, protein extract, cells (including tumorcells), tissues, polysaccharides, carbohydrates, fatty acids, teichiocacid, peptidoglycans, lipids, or glycolipids, individually or in anycombination thereof. An antigen also can include immunogenic fragmentsof nucleotides, polynucleotides, peptides, polypeptides, that can beisolated from the organisms referred to herein. An antigen in someembodiments is a cancer-specific molecule, such as a protein, peptide,lipid, nucleic acid, carbohydrate and the like.

Live, modified-live, and attenuated viral strains that do not causedisease in a subject can be isolated in non-virulent form or can beattenuated using methods known in the art, including serial passage in asuitable cell line or exposure to ultraviolet light or a chemicalmutagen. Inactivated or killed viral strains are those that have beeninactivated by methods known in the art, including treatment withformalin, betapropriolactone (BPL), binary ethyleneimine (BEI),sterilizing radiation, heat, and the like.

Two or more antigens can be combined to produce a polyvalent compositionthat can protect a subject against a wide variety of diseases caused bypathogens. Antigens can be combined in a single composition comprising acompound described herein, in some embodiments. In certain embodiments,a composition comprising multiple antigens is administered inconjunction with a separate adjuvant composition comprising a compounddescribed herein (e.g., concurrently or sequentially).

An immunostimulatory composition can include a microbe as an antigen(e.g., inactivated or attenuated bacteria, virus) or microbe component.Non-limiting examples of bacteria that can be selected includeAceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacilluspleuropneumoniae, Aeromonas hydrophila, Alicyclobacillus acidocaldarius,Arhaeglobus fulgidus, Bacillus anthracis, Bacillus pumilus, Bacillusstearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus,Bordetella bronchiseptica, Burkholderia cepacia, Burkholderia glumae,Campylobacter coli, Campylobacter fetus, Campylobacter jejuni,Campylobacter hyointestinalis, Chlamydia psittaci, Chlamydiatrachomatis, Chlamydophila spp., Chromobacterium viscosum,Erysipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichia canis,Escherichia coli, Haemophilus influenzae, Haemophilus somnus,Helicobacter suis, Lawsonia intracellularis, Legionella pneumophilia,Moraxellsa sp., Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasmamycoides subsp. mycoides LC, Clostridium perfringens, Odoribacterdenticanis, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida,Photorhabdus luminescens, Porphyromonas gu/ae, Porphyromonas gingivalis,Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris,Pseudomonas wisconsinensis, Pseudomonas aeruginosa, Pseudomonasfluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi,Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11-1,Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii,Rickettsia rickettsia, Salmonella typhimurium, Salmonella bongori,Salmonella enterica, Salmonella dublin, Salmonella typhimurium,Salmonella choleraseuis, Salmonella newport, Serratia marcescens,Spirlina platensis, Staphlyoccocus aureus, Staphyloccoccus epidermidis,Staphylococcus hyicus, Streptomyces albus, Streptomyces cinnamoneus,Streptococcus suis, Streptomyces exfoliates, Streptomyces scabies,Sulfolobus acidocaldarius, Syechocystis sp., Vibrio cholerae, Borreliaburgdorferi, Treponema denticola, Treponema minutum, Treponemaphagedenis, Treponema refringens, Treponema vincentii, Treponemapalladium, and Leptospira species, such as the known pathogensLeptospira canicola, Leptospira grippotyposa, Leptospira hardjo,Leptospira borgpetersenii hardjo-bovis, Leptospira borgpeterseniihardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae,Leptospira pomona, and Leptospira bratislava, and combinations thereof.

An inactivated virus, attenuated live virus, and/or portion of a virusmay be used in an immunostimulatory composition. Some examples ofviruses which can be used for antigen production include, but are notlimited to, Avian herpesviruses, Bovine herpesviruses, Canineherpesviruses, Equine herpesviruses, Feline viral rhinotracheitis virus,Marek's disease virus, Ovine herpesviruses, Porcine herpesviruses,Pseudorabies virus, Avian paramyxoviruses, Bovine respiratory syncytialvirus, Canine distemper virus, Canine parainfluenza virus, canineadenovirus, canine parvovirus, Bovine Parainfluenza virus 3, Ovineparainfluenza 3, Rinderpest virus, Border disease virus, Bovine viraldiarrhea virus (BVDV), BVDV Type I, BVDV Type II, Classical swine fevervirus, Avian Leukosis virus, Bovine immunodeficiency virus, Bovineleukemia virus, Bovine tuberculosis, Equine infectious anemia virus,Feline immunodeficiency virus, Feline leukemia virus (FeLV), NewcastleDisease virus, Ovine progressive pneumonia virus, Ovine pulmonaryadenocarcinoma virus, Canine coronavirus (CCV), pantropic CCV, Caninerespiratory coronavirus, Bovine coronavirus, Feline Calicivirus, Felineenteric coronavirus, Feline infectious peritonitis, virus, Porcineepidemic diarrhea virus, Porcine hemagglutinating encephalomyletitisvirus, Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV Type II,Porcine Reproductive and Respiratory Syndrome (PRRS) Virus,Transmissible gastroenteritis virus, Turkey coronavirus, Bovineephemeral fever virus, Rabies, Rotovirus, Vesicular stomatitis virus,lentivirus, Avian influenza, Rhinoviruses, Equine influenza virus, Swineinfluenza virus, Canine influenza virus, Feline influenza virus, Humaninfluenza virus, Eastern Equine encephalitis virus (EEE), Venezuelanequine encephalitis virus, West Nile virus, Western equine encephalitisvirus, human immunodeficiency virus, human papilloma virus, varicellazoster virus, hepatitis B virus, rhinovirus, and measles virus, andcombinations thereof.

Non-limiting examples of peptide antigens include Bordetellabronchiseptica p68, GnRH, IgE peptides, Fel d1, and cancer antigens, andcombinations thereof. Examples of other antigens include nucleotides,carbohydrates, lipids, glycolipids, peptides, fatty acids, and teichiocacid, and peptidoglycans, and combinations thereof.

Non-limiting examples of parasites that can be used for preparation ofantigens with an immunostimulatory composition include Anaplasma,Fasciola hepatica (liver fluke), Coccidia, Eimeria spp., Neosporacaninum, Toxoplasma gondii, Giardia, Dirofilaria (heartworms),Ancylostoma (hookworms), Trypanosoma spp., Leishmania spp., Trichomonasspp., Cryptosporidium parvum, Babesia, Schistosoma, Taenia,Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, andIsopsora, and combinations thereof. Also contemplated are externalparasites including, but not limited to, ticks, including Ixodes,Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, andHaemaphysalis species, and combinations thereof.

The amount of antigen used to induce an immune response can varyconsiderably depending on the antigen used, the subject, and the levelof response desired, and can be determined as known in the art. Forvaccines containing modified live viruses or attenuated viruses, atherapeutically effective amount of the antigen sometimes ranges fromabout 10.sup.2 Tissue Culture Infective Dose (TCID).sub.50 to about10.sup.10 TCID.sub.50, inclusive. For many such viruses, atherapeutically effective dose is sometimes in the range of about10.sup.2 TCID.sub.50 to about 10.sup.8 TCID.sub.50, inclusive. In someembodiments, the ranges of therapeutically effective doses are about10.sup.3 TCID.sub.50 to about 10.sup.6 TCID.sub.50, inclusive. Incertain embodiments, the ranges of therapeutically effective doses areabout 10.sup.4 TCID.sub.50 to about 10.sup.5 TCID.sub.50, inclusive.

For vaccines containing inactivated viruses, a therapeutically effectiveamount of the antigen sometimes is at least about 100 relative units perdose, and often in the range from about 1,000 to about 4,500 relativeunits per dose, inclusive. In some embodiments, a therapeuticallyeffective amount of the antigen is in a range from about 250 to about4,000 relative units per dose, inclusive, from about 500 to about 3,000relative units per dose, inclusive, from about 750 to about 2,000relative units per dose, inclusive, or from about 1,000 to about 1,500relative units per dose, inclusive.

A therapeutically effective amount of antigen in vaccines containinginactivated viruses also can be measured in terms of Relative Potency(RP) per mL. A therapeutically effective amount often is in the rangefrom about 0.1 to about 50 RP per mL, inclusive. In some embodiments, atherapeutically effective amount of the antigen is in a range from about0.5 to about 30 RP per mL, inclusive, from about 1 to about 25 RP permL, inclusive, from about 2 to about 20 RP per mL, inclusive, from about3 to about 15 RP per mL, inclusive, or from about 5 to about 10 RP permL, inclusive.

The number of cells for certain bacterial antigens administered in avaccine ranges from about 1.times.10.sup.6 to about 5.times.10.sup.10colony forming units (CFU) per dose, inclusive, in certain embodiments.In some embodiments, the number of cells ranges from about1.times.10.sup.7 to 5.times.10.sup.10 CFU/dose, inclusive, or from about1.times.10.sup.8 to 5.times.10.sup.10 CFU/dose, inclusive. In variousembodiments, the number of cells ranges from about 1.times.10.sup.2 to5.times.10.sup.10 CFU/dose, inclusive, or from about 1.times.10.sup.4 to5.times.10.sup.9 CFU/dose, inclusive, or from about 1.times.10.sup.5 to5.times.10.sup.9 CFU/dose, inclusive, or from about 1.times.10.sup.6 to5.times.10.sup.9 CFU/dose, inclusive, or from about 1.times.10.sup.6 to5.times.10.sup.8 CFU/dose, inclusive, or from about 1.times.10.sup.7 to5.times.10.sup.9 CFU/dose, inclusive.

The number of cells for certain parasite antigens administered in avaccine ranges from about 1.times.10.sup.2 to about 1.times.10.sup.10per dose, inclusive, in certain embodiments. In some embodiments, thenumber of cells ranges from about 1.times.10.sup.3 to about1.times.10.sup.9 per dose, inclusive, or from about 1.times.10.sup.4 toabout 1.times.10.sup.8 per dose, inclusive, or from about1.times.10.sup.5 to about 1.times.10.sup.7 per dose, inclusive, or fromabout 1.times.10.sup.6 to about 1.times.10.sup.8 per dose, inclusive.

Excipients

Aqueous immunostimulatory compositions can provide certain advantages.They are readily formulated and administered, and can induce few or lessserious injection site reactions. However, aqueous immunostimulatorycompositions with an antigen tend to diffuse from the injection site,are cleared by the subject's liver, and generate an undesirablenon-specific immune response.

Oil, when added as a component of an adjuvant, generally provides a longand slow release profile. Oils that can be utilized are metabolizableoils or non-metabolizable oils. An oil can be in the form of anoil-in-water, a water-in-oil, or a water-in-oil-in-water emulsion. Anoil-in-water emulsion can be provided in some embodiments, and can becomposed of an AMPHIGEN® formulation. This formulation comprises anaqueous component, lecithin, mineral oil, and surfactants. Patentsdescribing the components of the formulation include U.S. Pat. No.5,084,269 and U.S. Pat. No. 6,572,861. An oil component can be presentin an amount from 1% to 50% by volume, or in an amount of 10% to 45%; orin an amount from 20% to 40% in some embodiments.

Suitable oils can include alkanes, alkenes, alkynes, and theircorresponding acids and alcohols, the ethers and esters thereof, andmixtures thereof. Individual compounds of the oil often are lighthydrocarbon compounds, i.e., such components often have 6 to 30 carbonatoms. The oil can be synthetically prepared or purified from petroleumproducts. The moiety may have a straight or branched chain structure. Itmay be fully saturated or have one or more double or triple bonds. Somenon-metabolizable oils for use in the present invention include mineraloil, paraffin oil, and cycloparaffins, for example. A “light mineraloil” can be selected for use in an immunostimulatory composition. Onetype of oil utilized is obtained by distillation of petrolatum, and hasa slightly lower specific gravity than white mineral oil.

Metabolizable oils include metabolizable, non-toxic oils. This type ofoil can be any vegetable oil, fish oil, animal oil or syntheticallyprepared oil that can be metabolized by the body of the subject to whichan immunostimulatory composition is administered and is not toxic to thesubject. Sources for vegetable oils include nuts, seeds and grains.

Other components of an immunostimulatory composition can includepharmaceutically acceptable excipients, such as carriers, solvents, anddiluents, isotonic agents, buffering agents, stabilizers, preservatives,vaso-constrictive agents, antibacterial agents, antifungal agents, andthe like. Non-limiting examples of carriers, solvents, and diluentsinclude water, saline, dextrose, ethanol, glycerol, oil, and the like.Examples of isotonic agents include sodium chloride, dextrose, mannitol,sorbitol, lactose, and the like. Useful stabilizers include gelatin,albumin, and the like.

A surfactant can be used to assist in stabilization of an emulsion andcan be selected to act as a carrier for an adjuvant and/or antigen.Surfactants suitable for use include natural biologically compatiblesurfactants and non-natural synthetic surfactants, in some embodiments.Biologically compatible surfactants include phospholipid compounds or amixture of phospholipids. An example of a phospholipid isphosphatidylcholine (lecithin), such as soy or egg lecithin. Lecithincan be obtained as a mixture of phosphatides and triglycerides bywater-washing crude vegetable oils, and separating and drying theresulting hydrated gums. A refined product can be obtained byfractionating the mixture for acetone insoluble phospholipids andglycolipids remaining after removal of the triglycerides and vegetableoil by acetone washing. Alternatively, lecithin can be obtained fromvarious commercial sources. Other suitable phospholipids includephosphatidylglycerol, phosphatidylinositol, phosphatidylserine,phosphatidic acid, cardiolipin, and phosphatidylethanolamine. Thephospholipids may be isolated from natural sources or conventionallysynthesized.

Non-natural, synthetic surfactants that can be used include, withoutlimitation, sorbitan-based non-ionic surfactants, e.g.fatty-acid-substituted sorbitan surfactants (commercially availableunder the name SPAN® or ARLACEL®); fatty acid esters of polyethoxylatedsorbitol (TWEEN®); polyethylene glycol esters of fatty acids fromsources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g.,stearic acid available under the name SIMULSOL M-53®); polyethoxylatedisooctylphenol/formaldehyde polymer (TYLOXAPOL®); polyoxyethylene fattyalcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N),polyoxyethylene isooctylphenyl ethers (TRITON® X). In some embodiments,a surfactant, or combination of surfactants, is present in an emulsionin an amount of 0.01% to 10% by volume, sometimes 0.1% to 6.0%, and attimes 0.2% to 5.0%.

A pharmaceutically-acceptable carrier includes any and all solvents,dispersion media, coatings, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. Carrier(s) generally are compatible withother components of an immunostimulatory composition and not deleteriousto a subject when administered. A carrier often is sterile andpyrogen-free, and selected based on the mode of administration used, anda carrier utilized often is approved, or will be approved, by anappropriate government agency that oversees development and use ofpharmaceuticals. An immunostimulatory composition can include, incertain embodiments, a compatible pharmaceutically acceptable (i.e.,sterile or non-toxic) liquid, semisolid, or solid diluent that serves asa pharmaceutical vehicle, excipient, or medium. A diluent can includewater, saline, dextrose, ethanol, glycerol, and the like, for example.An isotonic agent can include sodium chloride, dextrose, mannitol,sorbitol, and lactose, among others. A stabilizer can include albumin,among others. An immunostimulatory composition can include, in someembodiments, an antibiotic or preservative, including, for example,gentamicin, merthiolate, or chlorocresol.

Preparation of Immunostimulatory Compositions

A compound described herein can be used in the manufacture of animmunostimulatory composition. Each dose can contain a therapeuticallyeffective amount of an antigen or antigens (e.g., vaccine) that can varydepending on the age and general condition of the subject, the route ofadministration, the nature of the antigen, and other factors. Theamounts and concentrations of other components in the immunostimulatorycomposition may be adjusted to modify the physical and chemicalproperties of the composition, and can be determined. Animmunostimulatory composition can be homogenized or microfluidized asdescribed hereafter.

An immunostimulatory composition can be prepared as an immunestimulating complex (ISCOM). An ISCOM can be prepared by combining asaponin, a sterol, and a phospholipid. For example, an ISCOM can contain5% to 10% by weight Quil A, 1% to 5% cholesterol and phospholipids, andthe remainder protein. The ratio of saponin to sterol in the adjuvantformulations sometimes is in the order of from 1:100 weight to weight(w/w) to 5:1 w/w. In some embodiments, excess sterol is present and theratio of saponin to sterol can be at least 1:2 w/w, or 1:5 w/w. Incertain embodiments, saponin is in excess in relation to the sterol, anda ratio of saponin to sterol of about 5:1 w/w is used. ISCOM andISCOMATRIX are commercially available (e.g., Isconova AB (Sweden)).

In some embodiments, CARBOPOL® is used in combination with DDA in anamount of at least 0.1 part by weight of CARBOPOL® per part by weight ofDDA. In certain embodiments, at least 0.5 part by weight of CARBOPOL®per part by weight of DDA is used. In various embodiments, at least 1part by weight of CARBOPOL® per part by weight of DDA is used. Thecombination of CARBOPOL® and DDA often forms a complex whereby the DDAtertiary amine functional group immunofunctionalizes the carboxylic acidside groups on the polymer. This complex allows for specific immunecells to target an antigen and adjuvant simultaneously and co-deliverthe antigen and adjuvant together at the optimal time and concentrationto the said cells.

In some embodiments, a compound described herein is not formulated witha specific carrier, and sometimes is formulated in an aqueous or otherpharmaceutically acceptable buffer for preparation of animmunostimulatory composition. In some embodiments, an immunostimulatorycomposition is presented in a suitable vehicle, such as for example,additional liposomes, microspheres or encapsulated antigen particles. Anantigen, if present in an immunostimulatory composition, can becontained within the vesicle membrane or contained outside the vesiclemembrane. Soluble antigens often are inside and hydrophobic or lipidatedantigens often are contained within the membrane.

An immunostimulatory composition can be made in various forms dependingupon the route of administration, storage requirements, and the like.For example, they can be made in the form of sterile aqueous solutionsor dispersions suitable for injectable use, or made in lyophilized formsusing freeze-drying, vacuum-drying, or spray-drying techniques.Lyophilized compositions can be reconstituted prior to use in astabilizing solution, e.g., saline or HEPES. Thus, an immunostimulatorycomposition can be used as a solid, semi-solid, or liquid dosage form.

Phosphate buffered saline (PBS) may be used as an aqueous buffer medium,where the pH of the buffer may be neutral or slightly alkaline orslightly acidic. Accordingly, the pH can be in a range of pH 6 to 8, anda pH of about 7.0 to about 7.3 can be used in certain embodiments. ThepH can be adjusted using a base (e.g., NaOH) or base (e.g., HCl) asneeded. Typical concentrations include from 1N to 10N HCl and 1N to 10NNaOH, for example. The strength of the buffer can be between 10 to 50 mMPO.sub.4 and between 10 to 150 mM PO.sub.4 in some embodiments. Incertain embodiments, a composition forms particles, for examplenanoparticles, of about 10 nanometers to about 1000 nanometers, andsometimes, a composition forms particles with a mean, average or nominalsize of about 100 nanometers to about 400 nanometers.

An immunostimulatory composition can be homogenized or microfluidized,in some embodiments. An immunostimulatory composition may be subjectedto a primary blending process, such as by passage one or more timesthrough one or more homogenizers, in certain embodiments. Anycommercially available homogenizer can be used for this purpose, e.g.,Ross emulsifier (Hauppauge, N.Y.), Gaulin homogenizer (Everett, Mass.),or Microfluidics (Newton, Mass.). In some embodiments, animmunostimulatory composition homogenized for three minutes at 10,000rpm. Microfluidization can be achieved by use of a commercialmicrofluidizer, such as model number 11OY available from Microfluidics,(Newton, Mass.); Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.); andRainnie Minilab Type 8.30H (Miro Atomizer Food and Dairy, Inc., Hudson,Wis.). These microfluidizers operate by forcing fluids through smallapertures under high pressure, such that two fluid streams interact athigh velocities in an interaction chamber to form compositions withdroplets of a submicron size. In certain embodiments, the formulationsare microfluidized by passage through a 200 micron limiting dimensionchamber at 10,000.+/−.500 psi.

Administration of Immunostimulatory Compositions

Dose size of an immunostimulatory composition can range from about 1 mLto about 5 mL, inclusive, depending on the subject and the antigen. Forexample, for a canine or feline, a dose of about 1 mL is typically used,while in cattle a dose of about 2-5 mL is typically used. However, animmunostimulatory composition can be formulated in a microdose, wheredoses of about 100 microliters can be used.

Non-limiting routes of administration for an immunostimulatorycomposition include parenteral, oral, oronasal, intranasal,intratracheal, topical, injection and intradermal. Any suitable devicemay be used to administer the compositions, including syringes,droppers, needleless injection devices, patches, pump, particles (e.g.,gold microparticles), electrotransduction, electroporation and the like.The route and device selected for use will depend on the composition ofthe adjuvant, the antigen, and the subject, as known in the art. In someembodiments, an immunostimulatory composition is administered byintravesical instillation.

An immune response can be monitored after an immunostimulatorycomposition is administered to a subject. Methods for assessing animmune response are known in the art, and include methods providedherein such as, for example, assaying antibody titer, either specific ornon-specific, and measuring serum cytokine levels In some embodiments,an antigen-specific immune response (e.g., antigen-specific antibodies,antigen-specific cytotoxic T-cells (CTLs)) is assessed after animmunostimulatory composition is delivered. In some embodiments, an IgG1and/or IgG2 (e.g., IgG2a) antibody response is induced. An immuneresponse can be assessed after an immunostimulatory composition isdelivered. In some embodiments, a composition described herein induceslittle to no side effects (e.g., splenomegaly) when administered to asubject.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1 Synthesis of4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzoicacid (compound 7)

This Example details methods by which compound A and SC12 can beprepared, and includes the following methods and data:

-   -   a method for preparation of        4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzoic        acid (compound 7) and its conjugation with        1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)    -   conditions for preparation of compound 7 and scale-up of the        preparation on multi-gram scale    -   a method of conjugation of compound 7 with        1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to obtain        compound A    -   a method for preparation of compound A on multi-gram scale    -   analytical methods for intermediates and for the conjugated        compound    -   stability study of compound A    -   a method for preparation of SC12, also known as SC12, a        conjugated derivative of 7 with        1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE))    -   stability study of SC12

The following schemes present an example of methods that may be used toprepare compound A and SC12. Other synthetic methods may be used toprepare compound A and SC12, examples of these other synthetic methodsare provided in FIGS. 20-23.

A. Preparation of4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzoicacid 7

2,6-dichloropurine (100 g, 0.53 mol) is charged in a four necked roundbottomed flask, 3 L, equipped with mechanical stirrer, oil bath,thermometer, dropping funnel, reflux condenser and nitrogen inlet.N,N-dimethylacetamide (1 L) is added, followed by solidbromomethyl-benzonitrile (114.6 g, 0.58 mol, 1.1 eqv.) and potassiumcarbonate (109.7 g, 0.79 mol, 1.5 eqv.). The mixture is vigorouslystirred and heated at 85-90° C. for 3 hrs, then it is allowed to cool toroom temperature and added with water (2 L). A yellow abundant solidimmediately is formed; the mixture is stirred for 30 min, then it isfiltered in a Buchner funnel, washed with water (2×200 mL) and ethylacetate and dried at 65° C. in vacuum until constant weight is observed(about 5 hs). Intermediate 2, batch CH730/2/1 is obtained as a paleyellow solid, with the following sample amount and purity: 160 g; 99% Y;90.2% HPLC purity. NMR and MS analysis conforms to the structure.

The reaction is scaled up and repeated starting from 600 g of2,6-dichloropurine. Intermediate 2 batch CH730/3/1 is obtained, with thefollowing sample amount and purity: 950 g; 98.3% Y; 92% HPLC purity.

Intermediate 2 (100 g, 0.33 mol) is charged in a four necked roundbottomed flask, 3 L, equipped with mechanical stirrer, oil bath,thermometer, dropping funnel, reflux condenser and nitrogen inlet. Drydimethylformamide (700 mL) is added, followed by ammonia solution 7 N inmethanol (100 mL, 0.66 mol, 2 eqv.). The mixture is vigorously stirredat room temperature. After 2 hours a brown solution is obtained, then anabundant solid precipitated. The mixture is further stirred for 12hours, then the solid is filtered on a Buchner funnel and washed withethyl acetate (200 mL). The product is dried at 65° C. in vacuum untilconstant weight is observed (about 6 hs). Intermediate 3 batch CH730/3/2is obtained as a whitish solid with the following sample amount andpurity: 66 g; 71% Y; 92.9% HPLC purity, NMR and MS analysis conforms tothe structure.

The reaction is scaled up and repeated on 900 g of intermediate 2.Intermediate 3 batch CH730/6/2 is obtained, with the following sampleamount and purity: 680 g; 77% Y; 91% HPLC purity.

A four necked round bottomed flask, 1 L, equipped with mechanicalstirrer, oil bath, thermometer, dropping funnel, reflux condenser andnitrogen inlet is charged with 2-methoxy ethanol (500 mL). Sodium (6 g,0.26 mol, 1.5 eqv) is added in small pieces at room temperature andunder Argon atmosphere. Intermediate 3 (50 g, 0.175 mol) is added in oneportion. The reaction mixture is stirred and heated to 100° C. for 6hours, then it is allowed to cool to room temperature. Water (1 L) isadded and the mixture is stirred at room temperature for 30 min. Thesolid is filtered on a Buchner funnel, washed with water (200 mL) anddried in vacuum at 65° C. until constant weight (about 8 hours).Intermediate 4, batch CH730/2/3 is obtained as a whitish solid with thefollowing sample amount and purity: 40 g; 70% Y; 95% % HPLC purity. NMRand MS analysis conform to the structure.

The reaction is scaled up and repeated on 550 g of compound 3.Intermediate 4 batch CH730/6/3 is obtained with the following sampleamount and purity: 532 g; 78% Y. 94% HPLC purity.

Intermediate 4 (100 g, 0.3 mol) is charged into a four necked roundbottomed flask, 2 L, equipped with mechanical stirrer, oil bath,thermometer, dropping funnel, reflux condenser and nitrogen inletDichloromethane (1.5 L) is added and the mixture is vigorously stirredat room temperature. Bromine (19 mL, 0.37 mol, 1.2 eqv.) is added dropwise at room temperature. After stirring for 8 hs, the solid is filteredand washed with dichloromethane (300 mL) to give crude compound 5 as ayellow solid. It is crystallized with acetone (500 mL) to giveintermediate 5 as a pale yellow solid with the following sample amountand purity: Batch CH730/3/4; 109 g; 88% Y. 82% HPLC purity.

The reaction is repeated on 150 g of compound 4; intermediate 5, batchCH730/4/4 is obtained; with the following sample amount and purity: 170g; 92% Y; 81% HPLC purity. A third preparation is made; intermediate 5,batch CH730/11/4 is obtained; with the following sample amount andpurity: 80 g; 91% HPLC purity.

A four necked round bottomed flask, 3 L, equipped with mechanicalstirrer, oil bath, thermometer, dropping funnel, reflux condenser andnitrogen inlet is charged with methanol (700 mL). Sodium (11.9 g, 0.52mol, 3 eqv.) is added in small pieces. Intermediate 5 (70 g, 0.17 mol)is added to the solution in one portion. The suspension is vigorouslystirred at refluxuntila clear solution is obtained (about 6 hours). Themixture is allowed to cool to room temperature, then water is added (500mL) followed by sodium hydroxide (34 g, 0.85 mol). The mixture is againheated to reflux for 8 hours, then it is cooled to room temperature.Concentrated hydrochloric acid is added (120 mL); a white solidprecipitates from the reaction mixture. After stirring for 1 hr, thesolid is filtered on a Buchner funnel. After drying in vacuum at 65° C.(about 8 hs), crude compound 6 (50 g) is obtained. It is suspended inacetonitrile (500 mL) and added with Sodium Iodide (Aldrich, 34 g, 0.23mol). After the drop wise addition of chlorotrimethylsilane (Aldrich, 29mL, 0.23 mol), the mixture is vigorously stirred and heated to 50° C.for 3 hours. After cooling to room temperature, a saturated solution ofsodium hydrogen carbonate is added, to obtain pH 6 into the reactionmixture. The solid precipitated is filtered on a Buchner funnel andwashed first with water (100 mL), then with methanol (50 mL). Crudecompound 7 is obtained as a pale yellow solid; batch CH730/18/6b, withthe following sample amount and purity: 40 g, HPLC purity 89%

It is crystallized twice with glacial acetic acid (600 mL each time).After drying in vacuum at 65° C. for 8 hours, compound 7 batchCH730/18/6c is obtained; with the following sample amount and purity: 34g; 55% Y from 5; 93.6% HPLC purity.

The reaction is repeated on 70 g of intermediate 5; compound 7, batchCH730/16/6b is obtained; with the following sample amount and purity: 38g; 61% Y; 92% HPLC purity. The reaction is repeated again on 30 g ofcompound 5; compound 7, batch CH730/21/6d is obtained; with thefollowing sample amount and purity: 18 g; 62% Y; 92.2% HPLC purity.

Acid 7 is not soluble in most of the common solvents (methanol, ethanol,dichloromethane, ethyl acetate, acetonitrile, acetone, chloroform). Manyattempts are made aimed to crystallize acid 7; dimethylformamide,dimethylformamide/water, DMSO/water, methanol, acetone are tested, butin all cases the product after crystallization has the same purity asbefore crystallization. Glacial acetic acid may be effective inenhancing the purity of 7. The purity is increased after the firstcrystallization, but it remains unchanged when the treatment isrepeated. The target value (98% HPLC) has not been achieved.

Preparation of Compound A

Many attempts are made aimed to prepare compound A with good yield andpurity. First, the direct coupling of acid 7 with DOPE is attempted(method A and method B). Then acid 7 is activated before coupling withDOPE (method C and method D). While reasonable results are obtained bothwith method A and method D, difficulties may arise during the work up ofthe reaction mixture and during the purification phase.

Method A: acid 7 (2.6 g, 7.2 mmol) is suspended in dry dimethylformamide(10 mL) under Argon atmosphere. HATU(O-7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluroniumhexafluorophosphate; 2.94 g, 7.6 mmol, 1.05 eqv.) is added in oneportion, followed by triethylamine (2 mL, 14.4 mmol, 2 eqv). The mixtureis stirred at room temperature for 15 min, then a solution of DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 5.37 g, 7.2 mmol, 1eqv.) in dry dichloromethane (150 mL) is added drop wise. The resultingsolution is stirred for 12 hours, until complete conversion of thereagents. The HPLC analysis shows that compound A is about 85% in thecrude reaction mixture. Dichloromethane is evaporated under reducedpressure and the residue is added drop wise to water (150 mL). A solidseparates from the reaction mixture. The attempt of filtration undervacuum may fail because the product is not crystalline and the filter isblocked.

At this point dichloromethane (150 mL) is added and the phases are leftto separate. A milky suspension forms and the separation of the twophases is not possible. In the event of failure of the filtration and ofthe extraction procedures, the solvents are completely removed bydistillation under vacuum and the residue is purified by flashchromatography, eluting with dichloromethane/methanol/acetic acid8/2/0.1. Compound A is obtained as a white amorphous solid with anexample of HPLC purity of 94.6% (0.5 g).

The reaction is repeated starting from 15 g of acid 7. The outcome ofthe reaction is similar to the previous run. The crude is purified bychromatography, but the target product is obtained with low yield (7.2g; 16% Y). When the silica gel used for the purification is washed withmethanol/acetic acid 7/3, the residual product is recovered. Itspurification is attempted again by chromatography. The purification bychromatography is effective on 1-2 grams scale; increasing the amount ofcompound A charged on the column, a great amount of product is retainedby silica gel and the recovery is low. The amount of methanol and aceticacid has to be increased and at this point the product is recoveredquantitatively, together with its impurities.

A crystallization technique also is attempted to purify compound A.Diethyl ether, hexane, acetone, acetone/water and other solvents aretested. Methanol is effective in lowering some impurities, but afterprolonged heating in methanol a new impurity is detected (up to 20%).

At this point, the reaction conditions are studied to minimizeimpurities in the reaction crude. Lowering the temperature results in abetter profile and compound A is obtained with 88% HPLC purity in thereaction mixture.

Method B: the reaction of acid 7 with DOPE is attempted usingdicyclohexylcarbodiimide (DCC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as coupling agents.In both cases no reaction occurs and the starting material is recoveredunchanged.

Method C: the activation of acid 7 is attempted with1-hydroxypyrrolidine in dichloromethane as solvent. Due to theinsolubility of 7 in dichloromethane, the reaction fails.

Method D: acid 7 (10 g, 0.028 mol) is dissolved in a mixture ofacetonitrile (60 mL) and dimethylsulfoxide (DMSO) (60 mL) at roomtemperature and under Argon atmosphere. Carbonyl diimidazole (4.55 g,0.028 mol, 1 eqv.) is added and the resulting solution is stirred for 1hr. A solution of DOPE (NOF Corp.>99%; 20.8 g, 0.028 mol, 1 eqv.) in drydichloromethane is added drop wise. The reaction mixture is stirred for16 hours, until complete conversion of the reagents. Acetonitrile isremoved by distillation in vacuum; water (200 mL) is added to theresidue; a white solid separated, but the filtration is not possible.The mixture is centrifuged for 30 min; the solvent is discarded andcompound A batch CH730/16/8 is obtained as a solid, 25 g. It is rapidlypassed through silica gel, eluting withdichloromethane/isopropanol/acetic acid 7/2/1 (CH730/16/8c, with thefollowing sample amount and purity: 21 g; 89.6% HPLC purity) then it istreated with methanol at room temperature for 30 min and filtered on aBuchner funnel. Compound A is obtained as a solid 19 g, HPLC purity94.5%. The reaction is repeated on 20 g of acid 7 with similar results.

Comparing methods A and D, similar results are obtained as far as yieldand purity of crude compound A, but the impurity profile is different.It is determined the purification of the sample obtained with method Aon multi-grams scale is not feasible. The coupling reaction between acid7 and DOPE is repeated several times and the isolation of compound Awith purity>90% is not readily accomplished.

Synthetic Processes

FIG. 20 shows examples of other synthetic process embodiments that canbe utilized for manufacturing certain compounds having a structure ofFormula A or Formula B. FIG. 20 specifically shows synthetic processesfor manufacturing compound A and SC12. These process embodiments includean intermediate having a structure of Formula A or Formula B except forthe hydroxyl moiety attached to the fused ring portion (8-hydroxyl) is a—O—(C1-C6 alkyl) moiety. This —O—(C1-C6 alkyl) moiety then is convertedto the hydroxyl moiety shown in Formula A or Formula B. The —O—(C1-C6alkyl) moiety sometimes is a —OCH3 moiety (i.e., —OMe moiety) as shownspecifically in intermediate 9 of FIG. 20. The —O—(C1-C6 alkyl) moietycan be converted to the hydroxyl moiety by a process known in the art,such as a TMSCl/NaI hydrolysis procedure (e.g., Carey, Advanced OrganicChemistry IV Ed.—Part B: Reaction and Synthesis page 163) and/or amethyl enol ether hydrolysis (e.g., Bioorganic & Medicinal Chemistry 12(2004) 1091-1099).

FIGS. 21 and 22 show further examples of synthetic process embodimentsthat can be utilized for manufacturing certain compounds having astructure of Formula A or Formula B. FIG. 21 and FIG. 22 specificallyshow synthetic processes for manufacturing compound A and SC12. Theseprocess embodiments includes an intermediate having a structure ofintermediate 7 in Scheme 1 shown previously except that the primaryamine moiety in intermediate 7 of Scheme 1 is a secondary amine havingthe structure —NH-(prot), where the prot moiety is a protecting group(e.g., intermediate 13 in FIG. 21 and intermediate 17 in FIG. 22). Theseprocess embodiments also include an intermediate having a structure ofFormula A or Formula B except that the primary amine moiety in Formula Aor Formula B is a secondary amine having the structure —NH-(prot) (e.g.,intermediate 14 in FIG. 21 and intermediate 18 in FIG. 22). Any suitableprotecting group known in the art can be utilized, and the protectinggroup sometimes is a tert-butoxycarbonyl (Boc) protecting group as shownby way of example in FIG. 21 (e.g., intermediates 13 and 14 in FIG. 21)or a benzyl protecting group as shown by way of example in FIG. 22(e.g., intermediates 17 and 18 in FIG. 22). Certain protecting groupsare suitable for producing compounds in which Rd and Re are saturatedalkyl moieties (e.g., Boc and benzyl) and certain protection groups aresuitable for producing compounds in which Rd and Re are alkyl moietiesthat include one or more unsaturations (e.g., Boc).

FIG. 23 shows examples of other synthetic process embodiments that canbe utilized for manufacturing certain compounds having a structure ofFormula A or Formula B. FIG. 23 specifically shows a synthetic processfor manufacturing SC12. This process embodiment includes an intermediatehaving a structure of intermediate 7 in Scheme 1 shown previously exceptthat the primary amine moiety in intermediate 7 is a secondary aminehaving the structure —NH-(prot), where the prot moiety is a protectinggroup (e.g., intermediate 17 in FIG. 23). This process embodiment alsoincludes an intermediate having a structure of Formula A or Formula Bexcept that the primary amine moiety in Formula A or Formula B is asecondary amine having the structure —NH-(prot) (e.g., intermediate 18in FIG. 23). This process embodiment further includes an intermediatehaving a structure of intermediate 6 in Scheme 1 shown previously exceptthat the primary amine moiety in intermediate 6 is a secondary aminehaving the structure —NH-(prot) (e.g., intermediate 21 in FIG. 23). Anysuitable protecting group known in the art can be utilized, and theprotecting group sometimes is a benzyl protecting group as shown by wayof example in FIG. 23.

In FIGS. 20-23, the number designations for the various compounds in thesynthetic scheme may not correspond with the numbers used in otherfigures, or the numbers used in this Example 1.

Sample Preparation for HPLC:

Intermediate no 5: in a 10 ml class A volumetric flask about 10 mg,accurately weighted, of sample were dissolved in methanol with somedrops of dimethyl sulfoxide (final concentration about 1 mg/ml).Intermediate no 7: in a 10 ml class A volumetric flask about 5 mg,accurately weighted, of sample were dissolved in methanol with somedrops of dimethyl sulfoxide (final concentration about 0.5 mg/ml).

Fragmentation of Compound A (Top Compound Shown Below)

X-Ray Diffraction (XRD)

It was determined the sample of compound A was amorphous.

Dry Weight and Chemical Composition (CHN)

The experimental values were in accordance with the structure ofcompound A.

Optical Rotation

Sample preparation: 20 mg of compound A was dissolved in chloroform andanalysed. [α]D=−37.08 (deviation on the analysis: 43%). It wasdetermined the high value of the deviation was likely due to theopalescent behavior of the solution.

The analysis was repeated dissolving 5 mg of compound A in chloroform.It was found [α]D=−8.7 (deviation on the analysis: 16%).

Solubility

The method reported in the European Pharmacopoeia 6.0 was used.

Compound A was not soluble in water.

Compound A was not soluble in acetonitrile.

Compound A was soluble in chloroform (100 mg/mL).

Compound A was soluble in DMSO.

The solubility of compound A in DMSO was determined using threedifferent batches of product:

-   -   CH730/16/8c (HPLC purity 59%)    -   CH730/16/8g (HPLC purity 71.5%)    -   CH730/23/8e (HPLC purity 95.6%)

Batch CH730/16/8g was tested according to the method described in E.Ph:DMSO was added in 0.1 mL portions to 103.7 mg of product and thesuspension was shaken with a Vortex instrument for 3 min after eachaddition. It was found that the solubility of compound A batchCh730/16/8g in DMSO was 259 mg/mL. The batches CH730/16/8c, CH730/16/8gand CH730/23/8e were then tested stirring each suspension for a longertime; 100 mg of each batch dissolved completely in 0.2 mL of DMSO afterstirring for 30 minutes. According to this method, the solubility ofcompound A in DMSO was 500 mg/mL.

Stability of Compound A

Some samples of compound A were retested by HPLC and it was found thattheir purity diminished in few days. The results are reported in Table Abelow:

TABLE A Sample: Test date: 20/05/09 Retest date: 12/06/09 Retest date:17/06/09 CH730/16/8c Purity: 89.6% Purity: 67.2% Purity: 58.9% ReportHPLC n.#0045 Report HPLC n. #0065 Report HPLC n. #0102 Sample: Testdate: 11/06/09 Retest date: 18/06/09 CH730/16/8g Purity: 81.4% Purity:71.5% Report HPLC n. #0062 Report HPLC n. #0103 Sample: Test date:10/06/09 Retest date: 16/06/09 CH730/22/8 Purity: 57.5% Purity: 44.8%Report HPLC n. #0060 HPLC report n. #0093

The samples which underwent degradation had been stored in acrystallization vessel at room temperature under natural light. Two mainimpurities at RRT 1.1 and RRT 1.2 were always present. As a consequence,it was determined that compound A was not a stable compound at roomtemperature and/or in the presence of light. At this point, a stabilitystudy was conducted on batch CH730/16/8g. The following conditions ofstorage were tested:

-   -   solid at room temperature (about 25° C.) and in presence of        light    -   solid at external temperature (28-35° C.) under the sun-light    -   solid at +4° C.    -   solution in chloroform at room temperature and in presence of        light    -   solution in chloroform at +4° C.    -   solution in chloroform at external temperature (28-35° C.) under        the sun-light

The results obtained are reported in Table B hereafter.

TABLE B Stability of CH730/16/8G Conditions Samples of storage t = 0 t =1 day t = 2 days t = 5 days t = 6 days t = 7 days t = 11 daysCH730/16/8G Solution in RRT = 1: RRT = 1: / RRT = 1: / RRT = 1: RRT = 1:CHCl₃, RT 81.4% 80.9% 79.3% 80.5% 82.0% and light RRT = 1.11: RRT =1.11: RRT = 1.10: RRT = 1.11: RRT = 1.16: 8.6% 8.7% 9.9% 8.7% 6.9% RRT =1.23: RRT = 1.24: RRT = 1.24: RRT = 1.27: RRT = 1.29: 4.2% 4.4% 4.2%3.9% 3.7% (Report n^(o) (Report n^(o) (Report n^(o) (Report n^(o)(Report n^(o) #62) #70) #96) #107) #111) Solution in RRT = 1: / / / / /CHCl₃, 80.3% external T RRT = 1.11: and light 8.0% RRT = 1.27: 3.2%(Report n^(o) #108) Solution in RRT = 1: / / / / / CHCl₃, T 80.7% about30° C. RRT = 1.11: without light 8.6% RRT = 1.27: 3.8% (Report n^(o)#109) Solid, / RRT = 1: / RRT = 1: / / external T 37.6% 35.4% and lightRRT = 1.06: RRT = 1.06: 9.2% 17.8% RRT = 1.11: RRT = 1.14: 27.9% 7.2%RRT = 1.26: RRT = 1.17: 14.8% 12.6% (Report n^(o) RRT = 1.28: #106) 5.5%(Report n^(o) #112)The solid samples were almost completely degraded after few days.Compound A appeared more stable in solution. Because the sample ofcompound A batch CH730/16/8g had a low purity at t=0, the stabilitystudy was repeated on a freshly prepared sample, batch CH730/23/8e. Theresults obtained are reported in Table C hereafter.

TABLE C Stability of CH730/23/8E Conditions Sample of storage t = 0 t =2 days t = 5 days t = 7 days t = 20 days CH730/23/8E Solution in RRT =1: RRT = 1: RRT = 1: RRT = 1: RRT = 1: CHCl₃, room 95.6% 95.5% 95.4%95.5% 94.4% temperature RRT = 1.24: RRT = 1.24: RRT = 1.23: RRT = 1.23:RRT = 1.19: and light 0.9% 0.9% 1.0% 1.1% 1.2% RRT = 1.28: RRT = 1.28:RRT = 1.29: RRT = 1.28: RRT = 1.26: 1.3% 1.3% 1.2% 1.2% 1.5% (Reportn^(o) (Report n^(o) (Report n^(o) (Report n^(o) (Report n^(o) 91) 92)98) 104) 113) Solution in RRT = 1: RRT = 1: RRT = 1: RRT = 1: CHCl₃, +4°C. 95.3% 95.3% 95.2% 93.9% RRT = 1.24: RRT = 1.23: RRT = 1.23: RRT =1.18: 1.0% 1.1% 1.1% 1.4% RRT = 1.27: RRT = 1.28: RRT = 1.28: RRT =1.25: 1.4% 1.3% 1.3% 1.4% (Report n^(o) (Report n^(o) (Report n^(o)(Report n^(o) 93) 99) 105) 116) Solution in RRT = 1: RRT = 1: RRT = 1:RRT = 1: CHCl₃, 94.2% 90.2% 89.0% 55.6% external RRT = 1.27: RRT = 1.13:RRT = 1.14: RRT = 1.08: temperature 1.2% 2.7% 2.5% 6.8% and light(Report n^(o) RRT = 1.27: RRT = 1.28: RRT = 1.16: 94) 1.2% 1.1% 1.6%(Report n^(o) (Report n^(o) (Report n^(o) 100) 106) 117) Solid, room RRT= 1: RRT = 1: RRT = 1: RRT = 1: temperature 94.6% 91.7% 91.8% 68.5% andlight RRT = 1.24: RRT = 1.22: RRT = 1.21: RRT = 1.08: 1.3% 1.7% 2.3%14.5% RRT = 1.27: RRT = 1.27: RRT = 1.28: RRT = 1.18: 2.1% 3.8% 3.8%3.9% (Report n^(o) (Report n^(o) (Report n^(o) RRT = 1.24: 95) 101) 107)6.7% (Report n^(o) 118) Solid, +4° C. RRT = 1: RRT = 1: RRT = 1: RRT =1: 95.6% 94.7% 94.9% 90.4% RRT = 1.24: RRT = 1.23: RRT = 1.22: RRT =1.18: 0.8% 1.2% 1.4% 3.1% RRT = 1.27: RRT = 1.28: RRT = 1.28: RRT =1.25: 1.3% 1.6% 1.5% 3.4% (Report n^(o) (Report n^(o) (Report n^(o)(Report n^(o) 96) 102) 108) 119) Solid, RRT = 1: RRT = 1: RRT = 1: RRT =1: external 94.1% 92.0% 92.0% 43.8% temperature RRT = 1.27: RRT = 1.27:RRT = 1.27: RRT = 1.09: and light 3.6% 4.9% 5.7% 27.2% (Report n^(o)(Report n^(o) (Report n^(o) RRT = 1.18: 97) 103) 109) 9.0% RRT = 1.23:12.1% (Report n^(o) 120)

HPLC analysis confirmed that compound A undergoes a rapid degradation atT>25° C. and in the presence of light. It was demonstrated that thecompound was more stable in solution.

HPLC-MS Analysis on a Stressed Sample of Compound A.

The sample of compound A batch CH730/16/8g, stored for six days atexternal temperature (28-35° C.; HPLC purity 35.4%; report n. #0112)under sun light, was analyzed by HPLC-MS and compared with freshlyprepared batch CH730/23/8e. The aim of this study was to demonstratethat the impurities detected by HPLC during the stability study were notan analytical artifact, but they were really formed by the action ofheat and light.

PLC method Instrument Agilent 1100 Column C18 XTERRA 2.1 × 150 mm, 3.5 mT (° C.) 50° C. lambda nm) 220 and 280 Flow (ml/min)  0.15 Analysis time(min) 40 Mobile phase A: methanol:isopropanol:water 50:20:30 + 0.1%formic acid B: methanol:isopropanol 50:50 + 0.1% formic acid ElutionGradient T (min) % A % B Gradient  0 100  0  5 100  0 30  0 100 40  0100 Retention time (min) Compound A about 33

Sample Preparation

Solution a: the sample was dissolved in dimethylsulfoxide-isopropanol20:80 in order to have a concentration of 2 mg/ml

Solution b: “solution a” was diluted 1:5 with methanol:isopropanol:water 50:20:30+0.1% formic acid

Example 2 Synthesis of SC12

Described hereafter is preparation of(2-(4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzamido)ethyl2,3-bis(oleoyloxy)propyl phosphate).

The HPLC-MS analysis carried out on compound A batch CH730/16/8gdemonstrated that the main impurity formed by the degradation of thecompound was the oxidated derivative. Acid 7 (compound 7) was conjugatedwith 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and itsstability was studied. The product is referred to as SC12. Compounds SC8and SC18 were synthesized in a similar manner, except that compound 7was conjugated with 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine or1,2-distrearoyl-sn-glycero-3-phosphoethanolamine, respectively, insteadof with DLPE.

A. Preparation of SC12

In this example of a method of preparing SC12, Acid 7 (compound 7)(batch CH730/18/6d; 1 g, 0.0028 mol) is dissolved in a mixture ofacetonitrile (6 mL) and DMSO (6 mL) at room temperature and under Argonatmosphere. Carbonyl diimidazole (455 mg, 0.0028 mol, 1 eqv.) is addedand the resulting solution is stirred for 1 hr. A solution of DLPE(>99%; 1.78 g, 0.0028 mol, 1 eqv.) in dry dichloromethane was added dropwise. The reaction mixture is stirred for 16 hrs, till completeconversion of the reagents. Acetonitrile is removed by distillation invacuum; water (200 mL) is added to the residue; a white solid separated.The solid is filtered on a Buchner funnel and washed with methanol (5mL). After drying in vacuum at 35° C., SC12 is obtained as a whitesolid; 1.8 g, Y 65%, HPLC purity 90.3%, HPLC report n. 0121.

B. Analytical Characterization of SC12

The structure of SC12 was confirmed by ¹H-NMR, ¹³C-NMR and MS.

C. Stability of SC12

A stability study was performed on SC12 and the following conditionswere tested:

-   -   solution in chloroform at room temperature (about 25° C.) and in        presence of light.    -   solid at room temperature and in presence of light.    -   solid at external temperature (28-35° C.) under the sun light.

The samples were analyzed at t=0 and after 20 days.

The results are reported in Table 1 below, and the main impuritiesformed with their RRT are listed.

TABLE 1 Conditions Samples of storage t = 0 t = 20 days CH730/2/13Solution in RRT = 1: 90.3% RRT = 1: 88.0% CHCl₃, RT RRT = 0.46: 5.7% RRT= 0.88: 6.2% and light RRT = 1.21: 0.9% RRT = 1.21: 5.0% (Report n^(o)0121) (Report n^(o) 0139) Solid, external RRT = 1: 84.0% T and light RRT= 0.88: 6.6% RRT = 1.21: 7.1% (Report n^(o) 0141) Solid, RT RRT = 1:87.8% and light RRT = 0.88: 6.2% RRT = 1.21: 4.8% (Report n^(o) 0141)

The data obtained show that SC12 was more stable than compound A.Furthermore, Table 2 shows the comparison between the stability ofcompound A and SC12 with respect to heat. Solid samples of each compoundwere maintained at 80° C. and analyzed by HPLC.

TABLE 2 After 2 hrs After 22 hrs Sample t = 0 at 80° C. at 80° C.Compound A 95% HPLC 73% HPLC / SC12 90% HPLC 90% HPLC 88% HPLC

Example 3 Potency of Compounds

The potency of five samples with TLR7 agonist activity in a PBMC modeland in the mouse macrophage cell line Raw264.7 was determined bymeasuring the dose-dependent stimulation of the pro-inflammatorycytokines IL-6 and TNF-alpha.

Methods and Experimental Setup

The study was setup in two models as outlined below:

-   -   Model 1: Five TLR7 agonists were tested in the mouse macrophage        cell line model Raw264.7. Endpoints were measurement of IL-6 and        TNF-alpha.    -   Model 2: Five TLR7 agonists were tested for potency in a PBMC        model. End-points were measurement of secreted IL-6 and        TNF-alpha.

Model 1

The potency of 5 TLR agonists was assessed in comparison with a positivecontrol (Imiquimod) in a Raw264.7 cell line. EC50 values were determinedfor control and each drug candidate for IL-6 and TNF-alpha secretion.

Method: Raw264.7 cells were grown according to conditions from thesupplier using RPMI media and 10% FCS. The cells were plated in 96 wellplates and treated with TLR7 agonists for 24 hours in 7 doses. Theconditioned media was removed after 24 hours for ELISA analyses. Thecells were subsequently assayed for viability using the XTT assayaccording to the guidelines from the supplier.

Experimental Setup:

-   -   1. Untreated cells    -   2. Imiquimod    -   3. TLR7 agonist 1    -   4. TLR7 agonist 2    -   5. TLR7 agonist 3    -   6. TLR7 agonist 4    -   7. TLR7 agonist 5

Compounds were tested in 7 doses (0,003-0,01,03-0,1-0,5-2,0-10,0micromolar). The experiment was performed in quadruplicate wells foreach testing series, and the supernatant pooled and measured by ELISA intriplicates.

Model 2

The potency of the 5 TLR agonists was assessed based on the ability tostimulate IL-6 and TNF-alpha secretion, and potency was compared with apositive control (Imiquimod) in a PBMC model. EC50 values weredetermined for control and each drug candidate for IL-6 and TNF-alphasecretion.

Method: PBMCs were purified from three donors and plated into 96 wellplates at 2×10⁵ cells/well in RPMI media including human 2% heatinactivated AB serum, glutamine, Pen-strep and beta-mercaptoethanol. Thecells were treated with TLR7 agonists for 24 hours in 7 doses. Theconditioned media was removed for ELISA analyses for IL-6 and TNF-alpha,and cell survival determined by the XTT method according to the protocolfrom the supplier.

Experimental Setup:

-   -   1. Untreated cells    -   2. Imiquimod    -   3. TLR7 agonist 1    -   4. TLR7 agonist 2    -   5. TLR7 agonist 3    -   6. TLR7 agonist 4    -   7. TLR7 agonist 5

Compounds were tested in 7 doses (0,003-0,01,03-0,1-0,5-2,0-10,0micromolar).

Data Handling:

Concentrations of IL-6 and TNF-alpha were determined by ELISA from R&DSystems using the Microsoft Excel Software. The results were analyzed inGraph Pad Prism in order to prepare dose-response curves, and fordetermination of EC50 values.

Statistical Analyses:

Due to the number of donors, statistical evaluation between individualEC50 values was not relevant.

Results and Discussion:

In Model 1, IL-6 and TNF-alpha secretions were induced dose-dependentlyby all compounds, but not by DMSO by itself (FIGS. 1-2). The compoundsreached different levels of maximum cytokine levels, and also showeddifferent EC50 values. The order of potency was as follows, with themost potent first: compound A <SC12<SC18<SC8=freepharmacophore<Imiquimod (Table 3). Free pharmacophore (“free ph”) hasthe structure:

The XTT assay (FIG. 3, where the Y-axis=relative survival, thex-axis=concentration for treatment) showed that the cells were slightlyaffected by the higher concentrations of compounds, which for somecompounds was reflected in a decreased cytokine production at thehighest concentrations (in particular for compound A, SC12 and SC18;FIGS. 1 and 2). Since the maximum plateau of the dose-response curveswas determined based on the cytokine levels induced by the highestconcentrations, this plateau could be slightly increased if the highestconcentration of the compounds was not considered. However, the doses inthe dynamic range would not be affected by this, which means that theEC50 values would be minimally affected.

TABLE 3 EC50 values determined in the Raw264.7 cell line on the basis ofthe 7 doses tested of each compound. Raw Free Compound 264.7 Imiquimodph A SC12 SC18 SC8 EC 50 IL 2.005 0.531 0.006 0.042 0.310 0.669 6 mM EC50 2.156 0.707 0.009 0.041 0.499 0.603 TNF-alpha

In conclusion for Model 1, compound A was the most potent TLR7 agonist,followed by SC12, SC18, SC8 together with free pharmacophore and finallyImiquimod. In this respect, all 5 compounds were more potent thanImiquimod in this model.

For Model 2, PBMCs were derived from buffy coats from healthy anonymousadult human donors. Both IL-6 and TNF-alpha secretions were induceddose-dependently by most compounds in the three donors. A few compoundslike Imiquimod, SC8 and the free pharmacophore showed weak ability toinduce the cytokines in some donors, where only the highest dose inducedcytokine production. Compound A was the most potent compound for IL-6secretion in all three donors. In donor 2, SC12 was as potent ascompound A, whereas SC12 was the second most potent compound in donor 1and 3, Table 4. On average, the compounds showed an order of potency asfollows: Compound A <SC12<free pharmacophore<Imiquimod<SC18<SC8. SC8showed levels of cytokines which did not allow a solid dose-responsecurve. For TNF-alpha secretion, SC12 was on average slightly more potentthan compound A, based on the results from the three donors, Table 4.The order of potency was as follows: SC12<compound A <freepharmacophore<SC18<SC8<Imiquimod, FIGS. 7-9. However, the data forImiquimod and SC8 were ambiguous, and only induced weak TNF-alphasecretion in all three donors. The survival assay showed an overall goodsurvival of the cells throughout the study at all concentrations tested,with no obvious cytotoxicity observed. One donor, number 2, who wastreated with SC12 had an increased survival response (FIG. 10) but didnot reflect a difference in cytokine response (FIGS. 5 and 8).

TABLE 4 EC50 values determined in PBMCs from three donors for the 6 TLR7agonists with indications of average potency. Com- Free pound Imiquimodph A SC12 SC18 SC8 IL-6 secretion Donor 1 6.348 3.239 0.4597 1.439 7.80811.02 MC50 mM Donor 2 1.994 2.671 0.7401 0.8642 12.35 11.31 MC50 mMDonor 3 4.643 2.443 0.2549 2.783 3.293 7.385 MC50 mM Average 4.328 2.7840.485 1.696 7.817 9.905 EC50 mM TNFa secretion Donor 1 3.071 3.1080.6443 0.9948 2.185 0 MC50 mM Donor 2 24.94 2.042 0.6651 0.2754 1-,549.543 MC50 mM Donor 3 22.39 1.544 0.6391 0.1207 1.958 0 MC50 mM Average16.800 2.231 0.650 0.464 4.894 9.543 EC50 mM

In conclusion for Model 2, compound A and SC12 were the most potent TLR7agonists. Compound A was the most potent stimulator of IL-6, and SC12was slightly more potent than compound A for TNF-alpha secretion. Theother compounds showed different abilities to induce IL-6 and TNF-alphafrom the PBMCs in the different donors, and their potency cannot begenerally ordered. Imiquimod and SC8 showed cytokine induction in thehighest concentration tested and low levels of secreted cytokine. ThusEC50 values cannot be determined for Imiquimod and SC8. SC18 and freepharmacophore showed similar responses for both IL-6 and TNF-alphasecretion, which reached higher levels than for Imiquimod and SC8 butwith higher values than for the Raw264.7 model.

The Raw264.7 cell line responded to all compounds tested, with compoundA being the most potent, followed by SC12. These two were followed bySC18, SC8 and free pharmacophore and showed similar potencies, withImiquimod showing the weakest induction of IL-6 and TNF-alpha.

The PBMC experiment showed compound A and SC12 as the two most potentTLR7 agonists, followed by SC18 and free pharmacophore, but with lowcytokine secretion measured after treatment with Imiquimod and SC8.

Example 4 Test for Potency of TLR7 Agonists from Two Different Batchesof Compound a and SC12 in Human PBMCs Aim

To determine the potency of 2 TLR7 agonists produced in two differentbatches in human PBMCs for induction of IL-6 secretion.

Methods and Experimental Setup

PBMCs were purified from two donors and plated into 96 well plates at2×10⁵ cells/well in RPMI media including human 2% heat inactivated ABserum, glutamine, Pen-strep and β-mercapto-ethanol. The cells weretreated with TLR7 agonists for 24 h in 4 doses. The conditioned mediawas removed for ELISA analyses for IL-6, and the EC50 value determinedfor each compound and each batch.

Experimental Setup:

-   -   1. Untreated cells    -   2. Untreated cells (vehicle control)    -   3. Imiquimod    -   4. Compound A (new batch#20289)    -   5. Compound A (old batch#CH730/25/8)    -   6. SC12 (new batch#20288)    -   7. SC12 (old batch#CH730/2/13D)

All compounds were tested in four concentrations (10-1-0,1-0,01micromolar). IL6 was determined after 24 h incubation in conditionedmedia by ELISA (IL6 gave in the last experiment the most comparabledose-response results).

Data Handling:

Concentrations of IL-6 were determined by ELISA from R&D Systems usingthe Microsoft Excel software. The results were analyzed in Graph PadPrism in order to prepare dose-response curves, and for determination ofEC50 values.

Statistical Analyses:

Due to the low number of donors, statistical evaluation betweenindividual EC50 values as not determined.

Results and Discussion

IL-6 was induced dose-dependently by all compounds, except Imiquimod,which was only active in inducing IL-6 at the highest concentration (10microM). A summary of the results is shown in Table 5 with indication ofEC50 values for the two donors tested (top two rows), and compared tothe values from the first experiments on the two compounds performed onthree donors (bottom three rows). The EC50 values for Imiquimod seemedto be somewhat higher in this present experiment compared to the lastexperiment. This can be explained by the storage at 4° C., andpotentially the heating procedure used to solubilize the compoundcompletely before use. SC12 showed similar EC50 values comparing thisexperiment with the previous experiment when testing batch CH730/2/13D.The old SC12 batch (CH730/2/13D) showed also similar EC50 valuescompared to the new batch (#20288). Compound A showed also similar EC50values in both this and the previous experiment when testing batch(CH730/25/8). The new compound A batch #20289, showed also similar EC50values compared to the old batch. No test for cell survival wasperformed since the last experiment showed no cytotoxic activity in theconcentrations tested.

TABLE 5 EC50 values determined in PBMCs from 5 different donors atdifferent time points, with different batches of SC12 and compound A.Compound A EC50 SC12 new SC12 old Compound A old values/microM Imiquimod20288 CH730/2/13D new 20289 Ch730/25/8 Donor 1 17.97 1.869 2.007 0.7210.734 Donor 2 18.45 0.947 1.034 0.521 0.392 D1 6.348 1.439 0.460 D21.994 0.864 0.740 D3 4.643 2.782 0.255

Conclusions

The two TLR7 agonists SC12 and compound A showed similar EC50 in thepresent experiment, indicating that they contain the same amount ofactive compound. This further indicated that the compounds (CH730/2/13Dand CH730/25/8) have not lost activity during the 5 months storage inDMSO at 4° C.

Example 5 Investigation of the Metabolic Stability of Compound a in Rat,Rabbit, Minipig and Human Plasma and Metabolic Profiling in Rabbit andHuman Plasma; Comparison of Compound a and SC12 Stability in HumanPlasma

Abbreviations:

2-Piperidinoethyl 4-amino-5-chloro-2-methoxybenzoate—M7319

Acetonitrile—ACN

Atmospheric Pressure chemical Ionization—APCI

Dimethylsulfoxide—DMSO Electron Spray Ionization—ESI

Formic acid—HCOOH

Liquid Chromatography/Mass Spectrometry—LC/MS Methanol—MeOH MultipleReaction Model—MRM Retention Time—R.T. Ultra Performance LiquidChromatography—UPLC Abstract

Stability of compound A* (another batch of compound A) was tested inrat, rabbit, minipig and human plasma, and metabolic profiling wasassessed in rabbit and human plasma. Compound A* was highly metabolizedby esterases in rabbit and human, and in a lesser extent in minipig andrat species. Metabolism was studied in rabbit at 30 and 120 min and inhuman at 60 and 300 min, keeping approximately constant the percentageremaining of the parent in the two species. Three metabolites were foundin rabbit and two of them in human.

In rabbit the major metabolites were the monoester and the acidmetabolite, whereas only traces of the di-hydrolized metabolite wereobserved. In human plasma only the first two major metabolites,previously detected in rabbit, were identified at the selected timepoints and the acid product was the predominant metabolite at 120 min.

This study found that in human and rabbit species a comparable profileof clearance and metabolism profile was found, with the formation ofonly two major metabolites where the rate limiting step was thehydrolysis leading to monoester formation, which rapidly converted intothe acid derivative.

In a second experiment performed in human plasma with a second batch ofcompound A (batch 20289) in comparison with SC12 suggested that SC12 wasmore stable than compound A, because it was not metabolized up to 120min, and more than 70% of the compound was still present at 300 min. Onthe contrary, compound A showed instability after 60 min incubation

Introduction

Hydrolytic enzymes present in plasma strongly contributed to themetabolism of compounds. Many drugs containing an ester bond were usedas prodrug to increase permeability or solubility or to decrease toxicsystemic effect. Esterases exist in many variety and species differencescan generally result from the existence of different types in biologicalmedia and differences in their substrate specificity. Additionallybioconversion can be affected by various factors such as age, gender anddisease.

Objective

The purpose of the assay was to compare stability, as percentageremaining of the parent, in several plasma species at different timepoints. Profiling of the major metabolites formed after incubation inplasma at 2 time points was carried out in rabbit and human plasma.

A second experiment with a different batch of compound A was performedby utilizing a different batch of human plasma and in comparison withSC12.

Plasma Stability and Metabolism Studies Materials

The following substances were obtained from the source indicated: ACNfrom J. T Baker, Germany, lidocaine, verapamil and M7319 fromSigma-Aldrich. HCOOH from Fluka. Deionized water from MilliQ apparatus(Millipore).

Plasma Samples were Obtained from the Source Indicated:

Rat plasma from Charles River, Calco, Italy. Minipig, human and rabbitplasma from Biopredic, Rennes, France.

Compound A2-(4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzamido)ethyl2,3-bis(oleoyloxy)propyl phosphate

Batch code: compound A* (First experiment), 20289 (Second experiment).

Storage Conditions: 4° C. as powder, —20° C. as stock solution in DMSO

SC12:2-(4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzamido)ethyl2,3-bis(oleoyloxy)propyl phosphate

Batch code: 20288

Storage Conditions: 4° C. as powder, store desiccated and away fromdirect light

Instruments

UPLC (Waters) interfaced with a Premiere XE Triple quadrupole (Waters)for clearance determination and UPLC (Waters) interfaced with Ion TrapHCTultra (Bruker Daltonics) for metabolic profiling.

Method

First experiment: Test compounds (50 mM DMSO) were diluted at the finalconcentration of 250 μM (in duplicate) with ACN. Plasma of differentspecies (1 ml) was spiked with 10 μl of 250 μM solution of the compoundand aliquots of 50 μl volume were taken at 0, 15, 30, 60, 120 min and 5hrs, and immediately quenched with 200 μl of a solution of Verapamil 250ng/ml (internal standard, I.S.) in ACN. A 10 μl of MeOH was added toimprove solubility. Samples were then centrifuged for 5 min at 13000 rpmand analyzed as reported below. Lidocaine and M7319 were used asreference standards and incubated as described above. The supernatantfractions were analyzed by LC/MS/MS. Zero-time incubations were used as100% values. Percent loss of substrate in incubations was determined toestimate the in vitro half life of the test compound.

Metabolism experiments were performed at 50 μM final concentration oftest compound and samples collected at two time points established inlight of the half life of the compound, and analyzed by LC/MS/MS afteraddition of ACN and internal standard.

Second experiment: Test compounds (5 mM in DMSO) were diluted at thefinal concentration of 250 μM with ACN-MeOH 1:1.

Human plasma (1.180 ml) was spiked with 20 μl of 250 μM solution of thecompound (4.16 μM final concentration) and aliquots of 50 μl volume weretaken at 0, 15, 30, 60, 120 min and 5 hrs, and immediately quenched with200 μl of a solution of Verapamil 250 ng/ml (internal standard, I.S.) inACN:MeOH 95:5. Samples were then centrifuged for 20 min at 3000 rpm at10° C. and analyzed as reported below. Lidocaine and M7319 were used asreference standards and incubated as described above. The supernatantfractions were analyzed by LC/MS/MS. Zero-time incubations were used as100% values.

Sample Analysis

Sample analysis for plasma stability determination (First experiment)

Samples were analyzed on a UPLC (Waters) interfaced with a Premiere XETriple Quadrupole (Waters).

Eluents were:

Phase A: 95% H2O, 5% MeOH, 0.1% HCOOH

Phase B: 5% H2O, 95% MeOH, 0.1% HCOOH

Column: Acquity BEH C8, 2.1×5 mm 1.7 um at 55° C.

Injection.Vol.: 5 μl.

A chromatographic method is reported below in Table 6.

TABLE 6 Chromatographic method for clearance determination Flow Time(min) (ml/min) % A % B 0 1 90 10 0.2 1 90 10 0.3 1 0 100 4.0 0.6 0 100

ESI pos, Capillary 3.4 kV, Extractor 5V, Source T 115° C., Desolvation T450° C. Cone Gas L/h 98, Multiplier 630 V.

In Table 7 the MRM transitions applied to compound A were reported.

TABLE 7 MRM transitions and parameters applied Collision Cone EnergyCompound Q1/Q3 (V) (V) compound A 1086.6/604.4 35 30 1086.6/385.2

Sample Analysis for Metabolic Profiling

The samples were analyzed using a Waters UPLC chromatographic systemcoupled with a Bruker Daltonics HCTultra® ion trap Mass Spectrometer.Before the analysis of the incubated samples, compound A was infusedmanually to understand parent fragmentation. Infusion was performed bydiluting a 50 mM solution in DMSO to 1 μM with ACN/MeOH 1/1. Samplesolution was infused into the ion trap source at a flow rate of 4ul/min.

Through a T-union 75 μl/min of H₂O/ACN 1/1+0.1% formic acid from theUPLC system was mixed with the flow of the compound solution tostabilize the flow rate and the signal.

The following conditions were applied to the Ion Trap: ESI positive,Capillary −4 KV, Cap Exit 164.3V, Skimmer 40V, Trap Drive 88.4, Neb. Gas70 psi, Dry Gas 10 l/min, Dry Temp 350° C.

Incubated samples were analyzed on a UPLC (Waters) interfaced with anIon Trap HCT ultra (Bruker Daltonics).

Eluents were:

Phase A: 95% H2O, 5% MeOH, 0.1% HCOOH

Phase B: 5% H2O, 95% MeOH, 0.1% HCOOH

Column: Acquity BEH C8 50×2.1 mm, 1.7 um at 55° C.

Injection.Vol.: 5 ul.

A chromatographic method is reported below in Table 8.

TABLE 8 Chromatographic method for metabolic profiling Flow Time (min)(ml/min) % A % B 0 1 90 10 0.2 1 90 10 7 1 0 100 12 1 0 100

Sample Analysis (Second Experiment)

Compound A and SC12 were analyzed using a Waters UPLC chromatographicsystem coupled with a Bruker Daltonics HCTultra® Ion Trap MassSpectrometer.

Eluents were:

Phase A: 95% H2O, 5% MeOH, 0.1% HCOOH

Phase B: 5% H2O, 95% MeOH, 0.1% HCOOH

Flow 0.6 ml/min. Column: Supelco, Discovery HS F5, 3.3 cm×2.1 mm; 55° C.

Injection Volume: 10 μl.

A chromatographic method is reported below in Table 9.

TABLE 9 Chromatographic method Time (min) % A % B 0 90 10 0.5 90 10 1 0100 3 0 100 3.1 90 10 3.5 90 10

The following conditions were applied to the Ion Trap:

-   -   For compound A: ESI positive, Capillary −4 KV, Cap Exit 164.3V,        Skimmer 40V, Trap Drive 88.4, Neb. Gas 70 PSI, Dry Gas 101/min,        Dry Temperature 350° C.    -   For SC12: ESI positive, Capillary −4 KV, Cap Exit 200V, Skimmer        49.5V, Trap Drive 85.0, Neb. Gas 70 PSI, Dry Gas 101/min, Dry        Temperature 350° C.

MRM transitions used for the quantifications were reported in Table 10.

TABLE 10 MRM transitions Compound [MH]+ Transitions Compound A 1085.6 603.6→385.23 SC12 921.6 439.45→385.23

Data Analysis

Stability was calculated as percentage remaining of the area ratiocompound/I.S. at each time point vs. area ratio compound/I.S. at time 0min. A general stability classification is reported in Table 11.

TABLE 11 General stability classification at 1 hr of incubation %remaining >80 80-60 60-30 <30 Classification Stable Slightly unstableUnstable Unstable

Metabolism was studied at 60 min and 300 min in human plasma, and at 30and 120 min in rabbit plasma, i.e. at the time points where the twospecies showed a similar percentage remaining of the parent compound.Assignment of the structures was done by comparison of the MS/MSanalysis of the spectra with the parent spectrum.

Results Plasma Stability (First Experiment)

Results obtained on plasma stability experiments are shown in Table 12.

Compound A was unstable in all the tested species; rabbit was thespecies with the highest clearance, followed by human and rat species;minipig showed the lowest clearance. In rabbit, rat and human plasma thefirst part of the curve up to 30 min is steep, whereas the remainingpart has a milder slope. Standards were in agreement with literaturedata.

TABLE 12 Percentage remaining of compound A in rat, rabbit, minipig andhuman plasma (% Remaining - Mean ± S.D.) Time (min) Rat Rabbit MinipigHuman 0 100 100 100 100 15 73.8 ± 4.5 38.7 ± 3.4  102.4 ± 3.3  99.7 (*)30 67.1 ± 8.8 10.2 ± 1.0  73.3 ± 5.0 40.4 ± 15.5 60 25.2 ± 0.5 7.4 ± 0.178.6 ± 2.9 36.1 ± 7.6  120 21.4 ± 1.9 4.4 ± 0.7 33.7 ± 2.3 17.4 ± 2.6 300 15.3 ± 1.0 1.9 ± 0.1 10.8 ± 0.3 7.8 ± 3.3 Data are expressed as Mean± S.D., n = 2, except when (*) where n = 1

Plasma Stability (Second Experiment)

Results on human plasma stability experiments for compound A and SC12were shown in Table 13. For compound A, an instability (about 80%remaining) was observed after 60 min incubation reaching a 56% remainingat 300 min. In this second experiment a different batch of compound Atogether with different analytical conditions and batch of human plasmain respect to the first experiment have been utilized: this couldexplain some differences observed between the percent remainingobtained.

SC12 was stable in human plasma up to 120 min. More than 70% of compoundwas still present after 300 min incubation. This data is in line withthat found in a previous experiment (data not shown). Standard compoundstested in the same experiment were in agreement with literature data(Table 14).

TABLE 13 Percentage remaining of compound A and SC12 in human plasmaTime Compound A SC12 (min) (Mean ± S.D.) % remaining 0 100 100 15 100.6± 14.5  112.6 ± 1.0 30 90.3 ± 13.3 110.0 ± 1.0 60 79.5 (*)  96.7 ± 6.3120 69.8 ± 16.8 102.1 ± 8.7 300 55.7 ± 9.9   72.4 ± 4.6 Data areexpressed as Mean ± S.D., n = 2; except when (*) where n = 1

TABLE 14 Percentage remaining of standard compounds in human plasma TimeLidocaine M7319 (min) (Mean ± S.D.) % remaining 0 100 100 60 107.1 ± 7.723.3 ± 1.4  300 105.1 ± 3.3 0.01 ± 0.01

Metabolic Profiling

Parent fragmentation: Major fragments were attributed as reported inTable 15 from MS/MS spectrum.

TABLE 15 Attribution of compound A major fragments Delta MH⁺ (m/z) m/zProposed Structure 1085 parent

603 −482

385.6 −700

327 −758

Metabolites Profiling

Metabolic profiling was studied in rabbit and human plasma. In bothmatrixes the parent compound (50 uM starting concentration) was totallymetabolized at the last time point. Metabolites were detected in Fullscan and peaks were assigned by MH+ and MS/MS spectra. The parentcompound showed a low response in Full Scan profile, therefore initialFull Scan chromatograms were not significant.

A summary of the metabolites with MH+ and retention time was reported inTable 16. In rabbit species three metabolites with MH+ of 557, 821 and360 respectively, were detected at retention times (r.t.) 1.1, 6.3 and6.4 min.

The most abundant peak corresponded to MH+821 (M2) that was assigned tothe mono-ester product which was converted 1:1 into the acid metaboliteat 120 min; only a small peak corresponding to the di-hydrolized productwas present (MH+557, M1). A similar profile was also observed in humanplasma where the two major metabolites showed the same MH+ and retentiontime of rabbit profile and their ratio was 1:1 after 60 min ofincubation, while metabolite M3 was the major product at 300 min. Tracesof di-hydrolized metabolite (MH+557) were present in human plasmaalready at time 0 and therefore it was not considered as metabolite.Therefore it was hypothesized that the rate determining step ofmetabolism was the formation of the monoester whereas all the otherdegradation steps occur in a much faster way. A potential selectivity ofhydrolysis in position 1 or 3 of the di-acyl glycerol moiety was notattributed.

TABLE 16 Identified metabolites in rabbit and human plasma M1 M2 M3 min1.1 6.3 6.4 MH+ 557 821 360 Rabbit x x x Human x x

Conclusions

Compound A stability in plasma was studied in four species: rabbit,human, rat and minipig. The product was highly metabolized by esterasesin rabbit and human and at a lesser extent in minipig and rat.Metabolism was studied in rabbit at 30 and 120 min and in human at 60and 300 min keeping approximately constant the percentage remaining ofthe parent in the two species. Three metabolites were found in rabbit(M1, M2 and M3) and two of them in human (M2 and M3). In rabbit themajor metabolites were the monoester and the acid metabolite whereasonly traces of the di-hydrolized metabolite were observed. In humanplasma only the monoester and the acid metabolite, previously detectedin rabbit, were identified at the selected time points and the acidproduct was the predominant metabolite at 120 min.

In conclusion the two species present a comparable profile of clearanceand metabolism profile with the formation of only two major metaboliteswhere the rate limiting step was the hydrolysis leading to monoesterformation which rapidly converted in to the acid derivate.

SC12 appeared more stable than compound A in human plasma, with a 70% ofcompound still present at 300 min.

Example 6 Potency of TLR7 Agonists in Human Whole Blood Assays onPlasmacytoid DCs, Myeloid DCs and B Cells Aim

To determine the potency of 2 TLR7 agonists in whole blood assays incomparison to Imiquimod. Specifically, it will be examined if the twoTLR7 agonists compound A and SC12 show differences in potency inactivation of immune cells in the whole blood assay. The optimalparameters in order to be able to show differences between thebiological effect of compound A and SC12 were believed to bemeasurements of B-cell, myeloid DC and plasmacytoid DC activation.

Methods and Experimental Setup

A volume of approximately 55 mL fresh whole blood was drawn inheparinized Vacutainers from three healthy adult anonymous volunteers asdescribed (J. A. Ida, Journal of Immunol Methods, 310, 2006, 86-99). Thedonors were healthy, did not suffer from known immune disorders, andwere not on medication. Before drawing the blood, the compounds wereadded to 96 well round bottom plates in a 10× diluted sample at 20 ul.The compounds were diluted in RPMI media without serum but withantibiotics. Antibiotics were added at a 10× concentration. Afterdrawing the blood, the whole blood sample was gently mixed to obtain ahomogeneous sample, and 180 ul was added to each well.

After 6 hours and 24 hours incubation, plasma was removed for ELISA(IL-6, IL-10, IL-12p40 and IFN-alpha). ELISA was made at the 6 and 24hours time point for all concentrations. After 24 hours incubation withselected compound concentrations, cells were analyzed for activationmarkers by FACS.

The following samples were prepared:

Experimental Setup:

1. Untreated cells2. Untreated cells (vehicle control)3. Imiquimod (old batch)4. Compound A (old batch)5. SC12 (old batch)

All compounds were setup in concentrations at0-0.01-0.03-0.1-0.5-2.0-10.0 micromolar as the final concentration.Vehicle control was DMSO control, where we used the highestconcentration used for the compounds. After addition of blood, allplates were gently agitated at 37° C. and 5% CO₂ until harvest. Thesamples were removed after 6 or 24 hours incubation, and pooled intoappropriate tubes (2 ml).

For the 6 hours time point, the plate was centrifuged 500×g. Supernatant(SN) was transferred to a tube and centrifuged at 10.000×g for 10 min toget rid of cells and protein aggregates. The clarified supernatant wasfrozen at −80 C until analysis.

For the 24 hours time point, the samples in the wells were pooled intotubes, which were centrifuged 500×g for 10 min at 4 C to clarify the SN.The SN was removed to another tube and centrifuged at 10.000×g for 10min. to get rid of aggregates etc. The clarified supernatant was frozenat −80 C until analysis.

FACS Analysis

A flow cytometric analysis was performed on whole blood from threedonors after treatment with two compound concentrations for 24 h. TheFACS analysis for donor 1 and donor 2 was made on another day than donor3. The compound concentrations were as follows:

B cell activation: 2 and 10 uMmDC/pDC: 0.1 and 0.5 uM

The FACS analysis was used to identify whether the test compounds couldinduce activation of B cells, and two different subsets of dendriticcells, namely the myeloid CD11c+/CD123− DCs and the plasmacytoidCD11c−/CD123+ DCs. The following markers were studied to identify theactivation status of the different subsets:

B-cells: HLA-DR/CD2O/CD40

pDCs: HLA-DR/CD123+/CD11c−/CD80HLA-DR/CD123+/CD11c−/CD86HLA-DR/CD123+/CD11c+/CCR7mDCs: HLA-DR/CD123−/CD11c+/CD80HLA-DR/CD123−/CD11c+/CD86HLA-DR/CD123−/CD11c+/CCR7

The FACS staining was performed according to the manufacturer'sinstructions. Lysis of red blood cells was performed before FACSstaining in order to minimize auto-florescence. To include as many Bcells and DCs in the study as possible the FACS analyses were performedon 500,000 cells in total for each staining. A P1 gate was set only toinclude relevant cells on FSC vs. SSC (see FIGS. 12-15). The results ofthe individual analysis were present as Mean Fluorescence Intensity

(MFI) values, for certain activation marker in a given gate setting,represent the actual cell subset. The isotype background values havebeen used to set the gates so that a maximum at 2% of unspecific stainedcells could be found in the positive gates.

Data Handling:

Concentrations of cytokines were determined by ELISA from R&D Systems.The results were analyzed in Graph Pad Prism in order to preparedose-response curves, and for determination of EC50 values. FACSanalysis was made by using Becton-Dickinson FACSDiva software andsubsequently illustrated in Graph Pad Prism.

Statistical Analyses:

Statistical evaluation between individual EC50 values was for relevantcytokines determined using two-tailed T-test with unequal variance.

Results and Discussion: Cytokine Secretion

IL-6 Secretion after 6 and 24 h

After 6 h incubation with compound A and SC12, all donors induced IL-6secretion in a dose-dependent manner. Imiquimod induced low andinsignificant amounts of IL-6, which did not allow a sigmoiddose-response curve as for compound A and SC12. EC50 values weredetermined for compound A and SC12 as seen in table 17. There was atendency for SC12 to show slightly more potent EC50 values than compoundA in all three donors. However, based on only three donors, this couldnot be confirmed as being statistically significant.

TABLE I7 EC50 values for IL-6 secretion after 6 hours incubation inwhole blood with TLR7 agonists. EC50-IL-6, 6 h Incubation/uM ImiquimodCompound A SC12 Donor 1 — 0.94 0.57 Donor 2 — 0.63 0.18 Donor 3 — 0.520.37

After 24 h incubation with compound A, SC12 and Imiquimod, all donorsinduced IL-6 secretion, but Imiquimod only in the highest concentrationtested. EC50 values were determined for all compounds, however, forImiquimod this determination was not accurate since only the highestconcentration was significantly above the detectable level, which didnot allow a sigmoid-dose response curve. The levels of IL-6 after 24hours incubation were only slightly above the levels of IL-6 seen after6 hours incubation. EC50 values of all compounds for IL-6 secretionafter 24 hours incubation was seen in table 18. There was the sametendency at 24 hours as for 6 hours, that SC12 was slightly more potent,and showed lower EC50 values than compound A in all three donors.Comparison between the result for all three donors on EC50 values forcompound A and SC12 using a two tailed T test showed a P-value of 0.07,which shows that the responses for the two compounds was notsignificantly different.

TABLE 18 EC50 values for IL-6 secretion after 24 hours incubation inwhole blood with TLR7 agonists. EC50-IL-6, 24 h Incubation/uM ImiquimodCompound A SC12 Donor 1 11.06 0.52 0.22 Donor 2 10.57 0.32 0.06 Donor 310.58 0.29 0.17

In summary, IL6-secretion was induced by compound A and SC12 in allthree donors to similar levels and with similar EC50 values, but SC12showed a tendency to be slightly more potent. Furthermore, the range ofIL-6 secretion (4000-8000 pg/ml) was in line with results published byClarke et al., Jour. Interferon & Cytokine Research, 29, 2, 2009,113-126.

IFN-Alpha Secretion after 6 and 24 Hours

After 6 hours incubation with compound A and SC12, all donors inducedIFN-alpha secretion in a dose-dependent manner, whereas Imiquimod didnot induce IFN-alpha secretion. Donor 1 and 2 induced IFN-alpha tolevels in the 2000 pg/ml range, whereas donor 3 only induced to the 500pg/ml range. EC50 values were determined for compound A and SC12 as seenin Table 19. There was again a tendency for SC12 to show slightly morepotent EC50 values than compound A in all three donors, however, thiswas not significant (P=0.23).

TABLE 19 EC50 values for IFN-alpha secretion after 6 h incubation inwhole blood with TLR7 agonists. EC50-IFN-alpha, 6 h Incubation/uMImiquimod Compound A SC12 Donor 1 — 1.17 0.98 Donor 2 — 1.84 0.83 Donor3 — 1.94 1.66

After 24 hours incubation with compound A, SC12 and Imiquimod, alldonors induced IFN-alpha secretion, but Imiquimod again only in thehighest concentration tested. EC50 values were determined for compound Aand SC12. The EC50 value for Imiquimod was again not accurate due toinduction at the highest concentration only. The levels of IFN-alphaafter 24 h incubation were below the levels of IFN-alpha seen after 6hours incubation for donor 1 and 2, indicating that this cytokine can beremoved by cells in the assay. EC50 values of all compounds forIFN-alpha secretion after 24 hours incubation were seen in table 20.After 24 hours all three donors induced IFN-alpha in the 1000 pg/mlrange, indicating that donor 3 responded to compound A and SC12 slowedthan donor 1 and 2. SC12 was again slightly more potent than compound A,although it was not significantly different (P=0.11).

TABLE 20 EC50 values for IFN-alpha secretion after 24 h incubation inwhole blood with TLR7 agonists. EC50-IFN-alpha, 24 h Incubation/uMImiquimod Compound A SC12 Donor 1 10.81 0.24 0.20 Donor 2 10.56 0.470.11 Donor 3 10.55 0.35 0.24

In conclusion, IFN-alpha secretion was induced by compound A and SC12 inall three donors to similar levels and with similar EC50 values, butSC12 showed a tendency to be slightly more potent. In comparison topublished results, the ranges of IFN-alpha secreted in this study(1000-2000 pg/ml) was higher than published data (<200 pg/ml), shownwith Resiquimod (Clarke et al., Jour. Interferon & Cytokine Research,29, 2, 2009, 113-126). However, this can be explained by expected lowerpotency of Resiquimod compared to compound A and SC12. Secondly,IFN-alpha secretion was known mainly to be induced by pDCs, and sinceIFN-alpha was secreted already after 6 hours in this study, it indicatesthat pDCs were activated as one of the initial responses seen aftertreatment of whole blood cells with compound A and SC12 (J. A. Ida,Journal of immunol methods, 310, 2006, 86-99).

IL-10 Secretion after 6 and 24 h

After 6 hours incubation with Imiquimod, compound A or SC12, and noIL-10 production was seen, indicating that IL-10 secretion was asecondary effect to treatment of whole blood cells with the TLR7agonists. After 24 hours incubation with compound A, SC12 or Imiquimod,all donors induced IL-10 secretion, but Imiquimod again only in thehighest concentration tested. EC50 values were determined for allcompounds, however, for Imiquimod this determination was again notaccurate due to induction of IL-10 at the highest concentration only.EC50 values of all compounds for IL-10 secretion after 24 hoursincubation were seen in table 21. After 24 hours all three donorsinduced IL-10 in the 2000-40000 pg/ml range. compound A and SC12 showedsimilar ability to induce IL-10, with no significant differences betweenthe two compounds.

TABLE 21 EC50 values for IL-10 secretion after 24 hours incubation inwhole blood with TLR7 agonists EC50-IL-10, 24 h Incubation/uM ImiquimodCompound A SC12 Donor 1 4.00 1.29 1.34 Donor 2 10.81 0.86 0.41 Donor 312.87 2.28 9.00

In conclusion, IL-10 was induced later in the assay, with no inductionafter 6 hours, but only after 24 hours incubation. This indicates thatIL-10 was induced as a secondary response in the assay, and possibly notas a direct effect of TLR7 ligation with the compounds tested. This wasconsistent with studies by Douagi et al, Journal of Immunology, 182,2009, 1991-2001, where IL-10 was induced in a human PBMC model onlyafter 12 and 20 hours, but not after 4 hours. Furthermore, Douagi et al,showed that mDCs were the main producers of IL-10 compared to pDCs.Supported by studies by Boonstra et al., Journal of Immunology, 177,2006, 7551-7558, who showed that mouse macrophages and mDCs producedIL-10 much more potently than pDCs after TLR ligation, the currentresults on IL-10 secretion by TLR7 ligation, indicates that thesecretion of IL-10 occurs in mDCs or potentially macrophages present inthe whole blood assay, potentially as a secondary response.

IL-12p40 Secretion after 6 and 24 Hours

After 6 hours incubation with compound A and SC12, all donors inducedIL-12p40 secretion in a dose response manner, whereas Imiquimod did notinduce IL-12p40 secretion, not even in the highest concentrations. Alldonors induced IL-12p40 to levels in the 8.000-12.000 pg/ml range. EC50values were determined for compound A and SC12 as seen in table 22. Thecompounds seemed to be equally potent in induction of IL-12p40, with nosignificant differences.

TABLE 22 EC50 values for IL-12p40 secretion after 6 hours incubation inwhole blood with TLR7 agonists. EC50-IL-12, 6 h Incubation/uM ImiquimodCompound A SC12 Donor 1 — 2.31 1.96 Donor 2 — 2.09 2.36 Donor 3 — 3.433.56

After 24 hours incubation with compound A, SC12 or Imiquimod, all donorsinduced IL-12p40 secretion to levels of IL-12p40 in the 20.000-25.000pg/ml range after compound A and SC12 treatment, whereas Imiquimod didnot induce IL-12p40 even in the highest concentrations. EC50 values weredetermined for compound A and SC12 as seen in table 23. Both compoundsshowed similar EC50 values, with no significant differences.

TABLE 23 EC50 values for IL-12p40 secretion after 24 hours incubation inwhole blood with TLR7 agonists EC50-IL-12p40, 24 h CompoundIncubation/uM Imiquimod A SC12 Donor 1 — 2.87 4.10 Donor 2 — 2.74 1.42Donor 3 — 4.03 14.17

In conclusion, IL-12p40-secretion was induced by compound A and SC12 inall three donors to similar levels and with similar EC50 values. Theinduction was seen both at 6 and 24 hours treatment, with increasedamounts at the 24 hours time point. In mouse cells, IL-12p40 is mainlyproduced by mDCs upon TLR ligation, compared to the production inmacrophages and pDCs (Boonstra et al., Journal of immunology, 177, 2006,7551-7558). If a similar pattern of IL-12p40 expression was seen forhuman cells, it indicates that compound A and SC12 follow a similaractivation profile in human mDCs.

Conclusions Regarding Cytokine Secretion

Compound A and SC12 were potent inducers of DC secreted cytokinesidentified in the supernatant from whole blood cell assays, whereasImiquimod was a weak inducer of these cytokines. This result wasexpected based on previous results in a PBMC model with the samecompounds. There was a tendency for SC12 to be slightly more potent ininduction of IL-6 and IFN-alpha, compared to the potency of compound A.However, with the number of three donors used, this could not bedemonstrated to be statistically significant.

For IL-10 and IL-12p40 secretion, compound A and SC12 were equallypotent based on this study. IL-10 was not produced after 6 hours, butonly after 24 hours incubation. IL-12p40 was induced both after both 6and 24 hours incubation.

Based on the current knowledge involving TLR7 activation of PBMCs andwhole blood assays, pDCs are known to be the main producers ofIFN-alpha. IL-6 was produced by both mDCs and pDCs, IL-10 was producedmainly by mDCs, and IL-12p40 mainly by mDCs. The production pattern ofIL-6 and IFN-alpha could indicate that SC12 was slightly more potent inactivation of pDCs than compound A. In assays with human primary cells,the presence and activation with TLR7 ligands of pDCs was required forstimulation of B-cell proliferation, and the production of IFN-alphafrom pDCs was known to be required for activation of B-cellproliferation and initiation of antibody production (Douagi et al,Journal of immunology, 182, 2009, 1991-2001, FIGS. 2 and 3).

In the previous model with testing of compound A and SC12 in the PBMCmodel, compound A showed a tendency to be slightly more potent than SC12in IL-6 secretion, however, this was not significant in the previousstudy either. A difference in potency of compounds in the two models canpotentially be explained by differences in chemical or physicalproperties of the compounds, since differences in lipophilicitypotentially will show a difference in partition into the cell pool ineach assay. The whole blood assay contains approximately a 50% cellvolume, due the presence of large amounts of red blood cells andplatelets. In contrast, a PBMC model contains much lower cell volume(<5%). In this regard, the large amounts of cells present in the wholeblood assay may work as a buffer for highly lipophilic compounds.

FACS Analysis B-Cell Analysis (FIG. 12):

The analysis of expression of the activation marker CD40 on B cells wasmade on double positive HLA-DR (MHC class II) (P4 gate) and CD20 cells(B cell marker) (P8 gate). FIG. 12 shows the results from the threedonors after treatment with the test compounds for 24 hours, includingthe MFI values for CD40 expression on double positive HLA-DR+/CD20+Bcells after 24 hours incubation with test reagents as indicated,performed on whole blood from three donors (D1-D3).

The activation marker CD40, shows an increased expression in all threedonors after treatment with the control compound Imiquimod in thehighest concentration (10 uM), in comparison with untreated or DMSOtreated cells. Both test compounds, compound A and SC12, induced CD40expression in donor 1 and donor 2 in all tested concentrations. However,in donor 3 only the highest concentration of the two test compoundsinduced CD40 expression compared to untreated cells. In all threedonors, compound A showed a weak tendency to stimulate a slightly higherCD40 expression than SC12 in all three donors, when tested at 10 uM.However, this tendency cannot be confirmed as statistically significantdue to the small number of donors.

DC Analysis (FIGS. 13-15):

The expression pattern of the co-stimulatory activation marker CD80 andCD86 and the chemokine receptor CCR7 was investigated on two differentsubsets of DCs. Samples for DC analyses on untreated cells from donor 1was lost, but a parallel sample which was untreated (DMSO) control cellsserved as similar control cells.

1. Myeloid Dendritic Cells (mDC):

The analysis of myeloid DCs was based on HLA-DR+/CD11c+/CD123− cells,thus all analyzed cells was included in the HLA-DR+(P3) gate andCD11c+/CD123− (Q4) gate (see gates in FIGS. 13-15).

It was found that Imiquimod induced a weak expression of CD80 in donor 1and 2, which was in contrast to donor 3 where the CD80 expression washigh. Both test compounds, compound A and SC12, induced a noteworthyCD80 expression in all three donors. In two out of the three donors (D1and D3), SC12 in the highest concentration (0.5 uM) showed a potentstimulation on CD80 expression, which was higher than the levels seenfor compound A. In donor 2, compound A stimulated CD80 expressionslightly more potent than SC12 in the highest concentration only.

Analyses of CD86 expression in mDCs, showed that untreated cells alreadyexpressed high levels of CD86 in all three donors, which was not anuncommon observation. However, compound A further stimulates theexpression of CD86 in all three donors. SC12 induces a weak CD86expression in donor 2 and 3, but not in donor 1. The lowestconcentration of both test compounds at 0.1 uM, showed most potently toinduce CD86 expression. This was in contrast to the CD80 expressionwhere the highest tested dose at 0.5 uM, were the most potentconcentration.

The chemokine receptor CCR7, which is a lymph node homing receptor, wasalso investigated on the mDCs. CCR7 expression showed a higher donor todonor variation than CD80 and CD86. For all donors, compound A inducedthe highest CCR7 expression, and for donor 1 and 3 Imiquimod also showedhigh CCR7 expression, which was not seen in donor 2. SC12 was for alldonors a less potent stimulator of CCR7 expression.

2. Plasmacytoid Dendritic Cells (pDC)

The analysis of pDCs was based on HLA-DR+/CD11c−/CD123+ cells, thus allanalyzed cells were included in the HLA-DR+(P3) gate andCD11c−/CD123+(Q1) gate. (FIGS. 13-15).

In the pDCs both compound A and SC12, shows a comparable effect at theCD80 expression pattern in all three donors. However, SC12 induces aslightly higher CD80 expression than compound A in all three donors,except for the lower concentration in donor 3.

Expression of CD86 in pDCs, shows that SC12 has a tendency to inducehigher CD86 expression than compound A in all three donors. However, themost potent concentration varies, as the lowest dose at 0.1 uM, inducesthe highest CD86 expression in donor 1 and 3, whereas 0.5 uM was themost potent concentration in donor 2. Imiquimod induce a similar highCD86 expression in donor 3 as SC12 and compound A.

The CCR7 expression pattern in pDCs was similar to what we found in themDCs. Compound A induced largely higher CCR7 expression than SC12 in allthree donors. Imiquimod in FIG. 16 shows MFI values for CD80, CD86 andCCR7 expression in HLA-DR+/CD11c−/CD123+pDCs after 24 hours incubationwith test reagents as indicated, performed on whole blood from threedonors (D1-D3).

Conclusion of FACS Analysis:

The overall conclusions on the B cell studies was that compound A at thehighest concentration (10 uM) stimulates slightly higher levels of thematuration marker CD40 than SC12 in all three donors.

For DC activation, SC12 was largely more potent than compound Aregarding stimulation of the activation marker CD80, which was the casefor both mDCs and pDCs (although a few exceptions were seen).

For the activation marker CD86 the results were a bit different ascompound A was slightly more potent than SC12 in mDCs, whereas SC12 wasslightly more potent than compound A in pDCs. However, the most potentconcentration for expression of CD86 varies between the donors.

Expression of the chemokine receptor CCR7, showed that compound A wasmore potent than SC12 in both DC subsets in most donors. As for the CD86expression, the most potent compound concentration for CCR7 inductionvaried between the donors.

Imiquimod generally showed to be potent only in donor 3 in B cells, mDCsas well as in pDCs, for the expression of all investigated markers.

Results

Compound A and SC12 were potent in induction of the cytokines IL-6,IL-10, IL-12p40 and IFN-alpha, and increased expression of maturationmarkers on both B-cells, pDCs and mDCs. The differences between thebiological effect of compound A and SC12 measured on these parameterswere not significant. However, if a larger number of donors were used, astatistically significant effect might be possible to show. Severaleffects showed borderline significance, and some maturation markersshowed a tendency to be induced more potently by one of the compounds.

The tendencies showed:

Compound A showed a tendency to be more potent than SC12 for thefollowing end-points.

1. Induction of the maturation marker CD86 on mDCs2. Induction of the migration receptor CCR7 for both mDCs and pDCs3. Induction of the B-cell activation marker CD40

SC12 showed tendency to be more potent than compound A for the followingend-points.

1. Induction of the maturation marker CD80 on both mDCs and pDCs2. Induction of the migration marker CD86 on pDCs3. Induction of the cytokines IL-6 and IFN-alpha

No tendencies could be seen at the level of IL-10 or IL-12p40 induction.

Based on these results, it can not be concluded that compound A and SC12behave significantly different when incubated with fresh human blood.

SC12 was slightly more potent than compound A in pDC activation, sinceCD80 and CD86 were induced more potently on pDCs by SC12, and the pDCcytokine IFN-alpha was induced more potently by SC12. In addition,compound A might be slightly more potent in B-cell activation, inparticular seen when tested at the 10 uM concentration.

Example 7 Preclinical Placebo-Controlled Efficacy Study with Imiquimodand SC12 in an Orthotopic Rat Bladder

The goal of this study was to evaluate the efficacy of a liquidformulation of Imiquimod (R-487 (1)) and SC12 in an orthotopic bladdercancer model in F344 rats. Four groups were compared: Imiquimod, SC12,vehicle and a placebo-group. After treatment, the animal well-being wasmonitored, and the response on rat-bladder and tumor was evaluatedhistopathologically.

Animals, Material & Methods Tumor Cells

The AY-27 rat bladder cancer cell-line was established from a primarybladder tumor in FANFT (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide)fed Fischer F344 rats. The cell-line was kindly provided by theUniversity of Alberta and Cross Cancer Institute, Edmonton, Alberta,Canada. The cells were cultured as a monolayer in RPMI-1640 (medium withL-glutamine (Invitrogen, Carlsbad, Calif.)), supplemented with 10% fetalcalf serum (Sigma-Aldrich, St. Louis, Mo.), 100 U/mL penicillin G and100 μg/mL streptomycin (Invitrogen, Carlsbad, Calif.) in a humidified95% air/5% carbon dioxide atmosphere. The medium was replaced two timesa week, and when confluent, the cells were passaged with standardtrypsinization procedures. Passage numbers used for the experiments were28 and 29.

Animals

A total of 56 female Fischer F344 rats were purchased from Charles River(L'Arbresle Cedex, France) and were acclimatized for at least one weekbefore the start of the experiment. The rats, weighing 170 g±10 g, werehoused in individual cages (Techniplast, Milan, Italy) with gold flakesbedding (SPPS, Frasne, France) and environmental enrichment, in atemperature controlled environment with a 12-hour light/dark cycle withfree access to standard chow and water. Each day, the rats were weighedand monitored for wellbeing. Animal procedures were performed accordingto protocols, which need to be approved by the Institutional Animal Careand Use Committee (IACUC), Committee for Animal Experiments (RadboudUniversity Nijmegen Medical Centre, The Netherlands) and in compliancewith Dutch and European regulations.

Sample Size Calculation

The group size was calculated using an expected therapy effect of 50%,an α of 0.05, a power of 80% and 80% tumor development. This resulted ina minimal group size of 14.

Tumor Cell Implantation

The tumor cell implantation was performed on day 0 according to theprotocol of Xiao et al (2). The F344 rats received isogenic tumor cells,resulting in a bladder tumor establishment of more than 80% (3).Enrofloxacin (Bayer, Leverkusen, Germany) (5-10 mg/kg) was injectedsubcutaneously for antibacterial prophylaxis before eachcatheterization. Experiments were performed under inhalation anesthesia:Isoflurane 2-5% (induction),followed by Isoflurane 2%, nitric oxide 0.5L/min and oxygen 1 L/min. The rat bladder was catheterized via theurethra with a 16-gauge (1.4 mm) plastic intravenous cannula (BDBiosystems, Erembodegem-Aalst, Belgium) and drained. The bladder waspre-conditioned with a 15 s instillation of 0.4 mL 0.1 M hydrochloride(HCl) and neutralized by adding 0.4 mL 0.1 M potassium hydroxide (KOH)for 15 s. The bladder was drained and flushed 3 times with 0.8 mL 0.01 MPBS. Freshly harvested AY-27 cells (passage 28 and 29) were resuspendedin medium. Immediately after bladder conditioning, and within 1 hourafter cell harvesting, the cells (1.5*10⁶ in 0.5 ml medium) wereinstilled in the rat-bladder and left indwelling for 1 hour. The ratswere rotated 90° every 15 minutes. After 1 hour, the catheter wasremoved, and the rats could void spontaneously.

TABLE 24 Treatment groups Group Intravesical instillation N 1 Imiquimod0.1% 14 2 SC12 0.38% 14 3 Vehicle 14 4 NaCl 14

Treatment

All the rats received an intravesical instillation on day 2 and 5. Firstthe rats were anesthetized by inhalation for one hour, as describedbefore. Subsequently the rats were catheterized via the urethra using a1.4 mm cannula (BD Biosystems), the bladder was drained and the pH wasmeasured using pH indicator strips (Merck, Darmstadt Germany). Theintravesical instillation was administrated using a 1 mL Luer-Loksyringe (BD Biosystems). Group 1 (n=14) was treated with 0.5 mlIMIQUIMOD 0.1%. Group 2 was treated with 0.5 ml SC12 0.38%. Group 3received an instillation with the vehicle (Phosal 50) and Group 4received an instillation with NaCl as a control. The testing agents weredissolved in Phosal 50 (Lipoid AG) as vehicle. The instillation remainedin the bladder for 1 hour, and the rats were rotated 90° each 15 min.After one hour, the catheter was removed. The pH of spontaneously voidedurine was measured using pH-indicator strips (Merck).

Pathological Evaluation

On day 12, the rats were sacrificed using carbon dioxide inhalation. Atnecropsy the internal organs were inspected and cystectomy wasperformed. The bladders were weighed, fixated using 4% buffered.laminated and embedded in paraffin. Sections of 5 μm were stained usinghematoxylin and eosin (H&E). A uro-pathologist evaluated the bladdersections, and scored the T stage using the TNM classification (UnionInternational Contre le Cancer, UICC, 2002). In addition, the totalnumber of tumors per bladder and the invasion depth of the tumors wasmeasured. The amount of inflammation in the bladder wall and/orsurrounding tissue was scored 0 (no inflammation), 1 (mild), 2(moderate) and 3 (severe inflammation).

Results

During the experiment, there were no signs of impaired wellbeing of therats and no rat reached a humane endpoint. An expected slight decreaseof weight was seen only the first day after anesthesia, but on the daysfollowing instillation, all rats regained weight. Mild hematuria on theday of catheterization was reported occasionally. On subsequent days,the urine turned normal. The pH of the urine before and after treatmentshowed no difference; the pH of all urines varied between 6.5 and 7.0.At necropsy no abnormalities to internal organs other than the bladderwere seen. At macroscopic evaluation tumor-positive bladders appear tohave tumor mass without extravesical growth. Only one rat bladder (group3, vehicle treated) showed a mass near the right urethral orifice,extending towards the right ureter.

Bladder Weight

The bladder weight correlated with the presence of tumor (p<0.0001,independent samples T test). In the table, an overview of the means andrange is given. There was a difference of mean bladder weight betweengroup 2 (SC12) and group 3 (Vehicle), (p=0.005, independent samples Ttest). No difference in mean bladder weight was seen between othergroups.

TABLE 25 Weight of the rat bladders per treatment and control group.Weight in grams. Group N Mean weight Standard deviation Range 1 140.1201 0.03488 0.0814-0.1891 2 12 0.1014 0.02258 0.0780-0.1422 3 120.1442 0.04163 0.0874-0.2028 4 13 0.1082 0.02311 0.0837-0.1587 Total 510.1184 0.03458 0.0780-0.2028

Inflammation

In almost all the rats a certain degree of inflammation was present.Between groups no statistically significant difference was observed(p=0.106, Pearsons Chi-square test). The mild and moderate degree ofinflammation accounted for 87.5% of all 56 cases.

Tumors and Tumor Response

The number of rats with a tumor positive bladder was 9 of 14 for theIMIQUIMOD treated group (group 1), 8 of 14 for the SC12 treated group(group 2), 11 of 14 for the vehicle-control group (group 3) and also 11of 14 for the NaCl control group (group 4). All tumors show a pT2 stage,except one pTa tumor in the vehicle-group. The pT2 tumors extend intothe detrusor muscle. In the rat with the pTa tumor, a small portion ofcancer cells were seen on top of the normal urothelial lining, with noinvasion. There was no statistically significant difference between theindividual groups in terms of tumor development. The difference in tumordevelopment between group 2 and the group 4 shows a non-significantp-value of 0.210 (Fischers Exact Test). The treatment given (e.g.IMIQUIMOD, SC12, vehicle or NaCl) was not predictive of the outcome(tumor positive versus tumornegative), when a logistic regressionanalysis was performed on the data.

Group Tumor free pTa Tumor pT2 Tumor Total 1 5 (35.7%)  9 (64.3%) 14(100%) 2 6 (42.9%) 1 (7.1%)  7 (50.0%) 14 (100%) 3 3 (21.4%) 11 (78.6%)14 (100%) 4 3 (21.4%) 11 (78.6%) 14 (100%)

Invasion Depth

Invasion depth of tumors was measured by a uro-pathologist onhistopathological evaluation of the H&E stained bladder sections. Thedeepest point at which tumor cells were seen, was taken as the invasiondepth. The mean invasion depth of tumor positive bladders did not showsignificant differences between individual treatment groups (independentsample T test, p=0.486-0.912), or between SC12-treated (groups 1,2) andcontrol treated (groups 3,4) animals (independent sample T-test p=0.705)

TABLE 27 Mean tumor invasion depth Group Mean invasion depth (mm)Standard deviation N 1 1.3333 0.45552 8 2 1.4250 0.70660 8 3 1.50000.56921 11 4 1.3909 0.55759 11

Tumor Number

The absolute number of tumors per bladder was counted by theuro-pathologist. The number of tumors in the vehicle control group washigher than the other groups. In univariate analysis, the number oftumors in the vehicle group (group 3) differed significantly from group1 and 4 (p=0.02) and from group 2 (p=0.006).

TABLE 28 Mean number of tumors per rat bladder Group Mean number oftumors Standard deviation 1 1.71 1.773 2 1.36 2.098 3 3.57 2.563 4 1.711.637

Conclusions

Imiquimod and SC12 cause a local immune response, that may lead toantitumor activity. No signs of toxicity were seen during thisexperiment. Although the effect of treatment on the tumor rate was notstatistically significant, a positive trend is seen towards theImiquimod and SC12-treated animals. Based on these data, futureexperiments may have an increased treatment frequency to improveefficacy.

REFERENCES FOR EXAMPLE 7

-   (1) Hayashi T, Crain B, Corr M, Chan M, Cottam H B, Maj R, et al.    Intravesical Toll-like receptor δ agonist IMIQUIMOD: Optimization of    its formulation in an orthotopic mouse model of bladder cancer 1.    International Journal of Urology 2010 May; 17(5):483-90.-   (2) Xiao Z, McCallum TJ, Brown KM, Miller GG, Halls SB, Parney I, et    al. Characterization of a novel transplantable orthotopic rat    bladder transitional cell tumor model 3. British Journal of Cancer    1999 October; 81(4):638-46.-   (3) Hendricksen K, Molkenboer-Kuenen J, Oosterwijk E, De Kaa CAHV,    Witjes JA. Evaluation of an orthotopic rat bladder urothelial cell    carcinoma model by cystoscopy. Bju International 2008 April;    101(7):889-93.

Example 8 Toxicity Analysis of SC12—Bacterial Mutation Assay

SC12 was examined for the ability to induce gene mutations in testerstrains of Salmonella typhimurium and Escherichia coli, as measured byreversion of auxotrophic strains to prototrophy. The five tester strainsTA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments wereperformed both in the absence and presence of metabolic activation,using liver S9 fraction from rats pre-treated with phenobarbitone andbetanaphthoflavone. SC12 was used as a solution in dimethylsulfoxide(DMSO). SC12 was assayed in the toxicity test at a maximum concentrationof 5000 micrograms/plate and at four lower concentrations spaced atapproximately half-log intervals: 1580, 500, 158 and 50.0micrograms/plate. Precipitation of SC12 was observed at the end of theincubation period at the two highest concentrations. No toxicity wasobserved with any tester strain at any dose level in the absence orpresence of S9 metabolism.

Using the plate incorporation method, SC12 was assayed at the maximumdose level of 5000 micrograms/plate and at four lower dose levels spacedby two-fold dilutions: 2500, 1250, 625, and 313 micrograms/plate. Notoxicity was observed with any tester strain at any dose level, in theabsence or presence of S9 metabolism. Precipitation of SC12 was observedat the end of the incubation period at the two highest concentrations.

As no increases in revertant numbers were observed at any concentrationtested, a pre-incubation step was included for all treatments of MainAssay II. SC12 was assayed at the same dose-range employed in Main AssayI. No toxicity was observed with any tester strain at any dose level, inthe absence or presence of S9 metabolism.

Dose-related precipitation of SC12, which did not interfere with thescoring, was observed at the end of the incubation period at the fourhighest concentrations.

SC12 did not induce two-fold increases in the number of revertantcolonies in the plate incorporation or pre-incubation assay, at any doselevel, with any tester strain, in the absence or presence of S9metabolism.

It was concluded that SC12 does not induce reverse mutation inSalmonella typhimurium or Escherichia coli in the absence or presence ofS9 metabolism, under the reported experimental conditions.

Example 9 Toxicity Analysis of SC12—Single Dose Intravenous Study inRats

The acute toxicity of SC12 was investigated after a single intravenousadministration to the Sprague Dawley rat followed by a 14-dayobservation period. A preliminary phase was carried out by subsequentlydosing groups of one male and one female rat at 76, 100, 85 and 90mg/kg, who were observed for a period of 7 days. An additional group,similarly composed, received the vehicle alone and acted as a control.No mortality occurred at 76 mg/kg. Clinical signs were limited topiloerection and reduced activity, observed on the day of dosing.

A second group was dosed at 100 mg/kg. Both animals died at dosing,after convulsions. A third group was then dosed at 85 mg/kg.Piloerection was observed on the day of dosing. A fourth group wasfinally dosed at 90 mg/kg. No mortality occurred. Piloerection wasobserved from Day 2 up to Day 4 of the study. No death occurred and noclinical signs were noted in male and female animals treated with thevehicle alone.

In the main phase, 5 male and 5 female animals were dosed at 90 mg/kgand observed for a period of 14 days. A second group, similarlyconstituted, received the vehicle alone and acted as control. Threemales treated at 90 mg/kg died immediately after dosing, while twofemales died at 2 hours post-dose. In addition, one male and one femaledosed at 90 mg/kg were found dead on Day 2 of the study. Twitches,ataxia, piloerection, reduced activity and hunched posture were themajor signs observed in animals before death. Ataxia, piloerection,reduced activity, hunched posture and semi-closed eyes were noted in thesurviving animals on the day of dosing. Piloerection was observed up toDay 3 of the study. A second group, similarly composed, was dosed at 80mg/kg. No mortality occurred and no clinical signs were recorded duringthe observation period. No mortality occurred and no clinical signs wereobserved in animals receiving the vehicle alone.

Surviving animals treated at 90 mg/kg and animals dosed at 80 mg/kgshowed a slight to moderate body weight loss on Day 2 of the study.Recovery occurred by Day 15 and the body weight changes were within theexpected range for this species and age of animals at the end of thestudy. No relevant changes in body weight were observed in animalsreceiving the vehicle alone during the study. Surviving animals werekilled at the end of the observation period with carbon dioxidenarcosis. All animals, including the early decedents, were subjected tonecropsy examination. No abnormalities were observed at necropsyexamination performed on all animals treated at 90 mg/kg (including theearly decedents), 80 mg/kg and on control animals. These resultsindicate that the test item SC12 induced mortality or significant signsof toxicity in rats following intravenous administration of a singledose at 90 mg/kg, while no mortality and no signs of toxicity wereobserved at 80 mg/kg. Therefore, the maximum tolerated dose in thisstudy was considered to be 80 mg/kg.

Example 10 Binding Assays

Imiquimod and SC12 were analyzed in enzymatic and radiologic bindingassays, as shown in FIGS. 17, and 18, respectively. SC12 and imiquimodwere evaluated in a radioligand binding assay among 73 primary moleculartargets using different human recombinant receptor types and subtypes ormembrane fraction from rodent tissue homogenates. SC12 was tested at afixed concentration of 30 microM.

Imiquimod was shown to bind to receptors that are associated withpain-related syndromes (e.g. adenosine and sodium channel), which arethe most common adverse events in patients after treatment with Aldara(imiquimod). SC12 did not bind this type of receptors.

Example 11 Investigation of the Intravenous Pharmacokinetics of SC12 inthe Mouse

The pharmacokinetics of SC12 was assayed in fasting male CD-1 mice.

Materials and Methods

An IV bolus was administered into the caudal vein at a dose of 1 mg/kgin 5 ml. The compound weight was 2.08/10.04 ml. 24 mice were studied.Sampling was obtained by exsanguination under anesthesia, at 5, 15, 30,60, 240, and 480 minutes, and at 24 hours. SC12 was administered in aformulation of 3% DMSO, 20% beta-cyclo-dextrin, in water, at a dosevolume of 5 ml/kg. Animals were sacrificed using ethyl ether at theconclusion of the experiment.

Sample preparation: In a Sirocco filter plate, 100 microliters of plasmawere added to 300 microliters of ACN/MeOH spiked with 5 microliters ofIS (IV298 10 micrograms per ml) plus 10 microliters of 5% H₃PO₄. Theplate was shaken for 10 minutes at 80 rpm and then filtered under vacuumfor 5 minutes.

Analytical method: LC/MS/MS: Premiere XE, Eluent: water (A) MeOH (B)plus 0.1% HCOOH gradient. 15% B to 100% B from 0.1 to 0.5, thenisocratic 100% B up to 1.5 minutes, flow 0.8 ml/min; Column Acquity UPLCBEH C18 1.7 microm 2.1×50 mm, injection volume of 5 microliters in a Tcolumn at 50° C. ESI positive, MRM, Extractor 5V; Capillary 3.5 kV; Tsource 115° C.; T desolv. 450° C. SC12: MH+921.5>385.05/439.29 CV35 CE33LLOQ: 5 ng/ml.

Data analysis: Non-compartmental analysis WinNonlin 5.1; lineartrapezoidal, uniform weight.

Results

No adverse behavioral effects were noted in the treatments.

SC12 shows a Cmax in plasma of 541 ng/ml with a short MRT, which isreflected in a high clearance (Table 29). Some inter-animal variabilitywas observed at the first time point (5 min), possibly due to the veryrapid clearance in the first part of the decay, whereas, in the secondpart of the curve, the concentration in plasma decreased slowly, beingunder the LLOQ (lowest limit of quantification) after 2 hrs.

Raw data and non-compartmental analysis output are reported in Table 30.

TABLE 29 Pharmacokinetic parameters T½ (min.) 130 Tmax (min.) 5 Cmax(ng/ml) 541 C0 (ng/ml) 2664 Tlast (min) 120 Clast (ng/ml) 6 AUClast(min * ng/ml) 11744 AUC INF obs (min * ng/ml) 12878 CI (ml/min/kg) 77.7MRT (min) 32 Vss (L/kg) 2.5

TABLE 30 Raw data and non-compartmental analysis output Time Plasmaconcentration (min) Mean ± S.D. (ng/ml) 5 540.70 ± 179.99 15 22.27 ±3.16  30 10.00 ± 1.68  60 7.62 ± 0.72 120 6.05 ± 0.78 240 <LLOQ 480<LLOQ 1440 <LLOQ

Example 12 Inhibition of Human CYP450 In Vitro

The interaction of SC12 with cytochrome P450 enzymes was tested usingFluorescent High Throughput P450 assays (Gentest); The IC50s of thecompounds was calculated on isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2C8,CYP2B6, CYP2D6, CYP2E1, CYP3A4 and CYP3A5).

Materials and Methods

Inhibition of the P450 isoforms was measured in specific assays usingspecific substrates that become fluorescent upon CYP metabolism.Compounds, dissolved in ACN (acetonitrile) (CYP2E1, CYP2C8, CYP2B6,CYP3A5) or DMSO (all remaining isoforms), were tested in duplicate (n=2)in concentration-response curves (eight concentrations) in a 96-wellplate containing incubation/NADPH regenerating buffer. Specificisoenzymes and substrates were added and incubated at 37° C. Reactionswere terminated at different times, depending on the assays, and platesread on a Fluoroskan Ascent at the appropriate emission/excitationwavelengths. Concentration-response curves performed in duplicate forknown inhibitors for each isoenzyme were tested in every assay aspositive control.

Data Analysis

For each compound and standard the IC50 (concentration at 50%inhibition) was determined by using Grafit v. 5.0.1.

Results Results are Shown in Table 31 (Compounds) and Table 32(Standards).

SC12 showed a moderate inhibition on CYP2E1, CYP3A5 and CYP3A4 isoformsand a weak inhibition on CYP2C9, whereas it did not appear to inhibitthe others isoforms activity. Because SC12 showed a low solubility,especially in ACN, results could be underestimated. The standardreference inhibitors in all the experiments performed showed theexpected potency.

TABLE 31 P450 results CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A5 CYP2E1CYP2CB CYP2B6 CEC MFC CEC AMMC BFC BFC MFC DBF EFC COMPOUND IC₅₀micromolar (Mean ± S.D.) SC12 >100 33.4 ± 0.3 >100 67.0 ± 3.8 13.6 ± 0.710.5 ± 0.5 9.2 ± 0.1 >100 >100 Abbreviations: BFC:7-Benzyloxy-4-(trifluoromethyl)-coumarin CEC: 3-Cyano-7-EthoxycoumarinAMMC: 3-[2(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarinDBF: Dibenzylfluorescein DMSO: Dimethylsulfoxide EFC:7-Ethoxy-4-trifluoromethylcoumarin MFC:7-Methoxy-4-trifluoromethylcoumarin

TABLE 32 P450 results on standard inhibitors Experiment data IsoformsStandard inhibitors IC50 (microM) CYP1A2 Furafylline 1.9 ± 0.1 CYP2C9Sulfaphenazole 0.27 ± 0.01 CYP2C19 Tranylcypromine 5.0 ± 0.2 CYP2D6Quinidine 0.009 ± 0.001 CYP3A4 Ketoconazole 0.012 ± 0.001 CYP3A5Ketoconazole 0.12 ± 0.10 CYP2E1 DDTC 0.85 ± 0.01 CYP2C8 Quercetin 3.5 ±0.4 CYP2B6 Tranylcypromine 6.9 ± 1.2

Example 13 Investigation of Metabolic Stability and Profiling ofCompound a in Mammalian Plasma, and Comparison of Compound a and SC12Stability in Human Plasma

The stability of compound A was tested in rat, rabbit, minipig and humanplasma up to 5 hours, and metabolic profiling was assessed in rabbit andhuman plasma. Compound A was highly metabolized by esterases/amidases inrabbit and human, and in a lesser extent in minipig and rat species.Metabolism was studied in rabbit at 30 and 120 min and in human at 60and 300 min, i.e. operating at approximately the same residualpercentages of the parent in the two species. Three metabolites werefound in rabbit and two of them in human.

In rabbit the major metabolites were the monoester (loss of one oleicacid, M2) and the acid metabolite (amide hydrolysis, M3), whereas onlytraces of the di-hydrolyzed metabolite (loss of both oleic acids, M1)were observed. In human plasma only the first two major metabolites,previously detected in rabbit, were identified at the selected timepoints and the acid product (M3) was the predominant metabolite at 120min.

In conclusion, in human and rabbit species a comparable profile ofclearance and metabolism profile was found, with the formation of onlytwo major metabolites where the rate limiting step is the hydrolysisleading to monoester formation (M2), which rapidly converted into theacid derivative (M3).

In a second experiment performed in human plasma with a second batch ofcompound A in comparison with SC12 suggested that SC12 is little morestable than compound A, because it was not metabolized up to 120 min,and more than 70% of the compound was still present at 300 min (compoundA unchanged at 300 min: 55%).

Example 14 Direct Proapoptotic Effects of SC12 on Skin Cancer CellLines: a Comparison with Imiquimod

In addition to its immunomodulatory effects, Imiquimod has been reportedto directly induce apoptosis in tumor cells, which has been confirmed intumors of different origin in vivo. The proapoptotic activity ofImiquimod may contribute to the antitumoral effects of Imiquimod invivo, as the required concentrations are still approximately 3 logsbelow the concentration in Aldara 5% cream.

Experimental Methods and Results Cell Lines:

Cutaneous Squamous Cell Carcinoma (SCC) cell lines (human) SCL-I,SCL-II, SCC-12, SCC-13 have been well characterized with regard to theirgrowth behavior and apoptosis sensitivity to death ligands (CD95L,TRAIL, TNF-alpha) as well as to other treatments. SCC cells are grownunder standard conditions (10% FBS).

Determination of the Direct Proapoptotic and Cytotoxic Effects of SC12on SCC Cells:

Time and dose dependency of effects on total cell numbers have beeninvestigated by real-time cell analysis, which is based on continuousmonitoring of electric conductance in microtiter wells (E-plates,Roche), which corresponds to the cell numbers. Different concentrationsof SC12 as well as Imiquimod (Imq) have been used to treat the 4 celllines and the cell numbers have been compared to untreated control cells(2 independent experiments for each cell line, triple values, differentconcentrations). For these assays 4 cell lines should be used to obtaina representative overview on the effects on SCC cells. Thus, cells havebeen treated with different concentrations of SC12 or Imiquimod; growthand apoptotic effects have been monitored by microscopy for at least 7days.

FIG. 17 shows reduced cell numbers with SC12 and Imiquimod. CutaneousSCC cell lines were continuously monitoring of electric conductance inmicrotiter wells (E-plates, Roche), which corresponds to the cellnumbers. TMX indicates SC12 in the charts. FIG. 18. provides photographsshowing similar morphological changes induced by SC12 and Imiquimod. Atday 3, cell detachment, morphological changes and inhibition ofproliferation can be observed in SCC cells treated with either SC12 orImiquimod. Time of treatment 3d, Concentrations: 120 microM.

Example 15 Compound A and SC12 as Adjuvants

A pilot immunization study was conducted using compound A and SC12adjuvanted protein antigens. The immunization experiments were performedwith two recombinant proteins expressed in E. coli. One antigen wasderived from the malaria parasite Plasmodium falciparum and the otherone from Mycobacterium ulcerans, which cause the ulcerative skin diseaseBuruli ulcer.

Groups of five mice received, with three week intervals, threesubcutaneous immunizations with 20 micrograms of target antigen mixedwith 10 nMol compound A or SC12.

After the third immunization, all 20 immunized mice had developed IgGresponses against the respective target antigens. The performance ofcompound A and SC12 was comparable. No local side effects, such asswelling or ulcerations were observed. A parallel immunization with acommercial adjuvant approved for use in mice yielded higher antibodytiters; but here local reactions were observed.

FIG. 19 shows the development of IgG titers against the M. ulceransantigen, (left: compound A, right, SC12).

Example 16 Investigation of the Exposure of Imiquimod and SC12 in MouseSerum after Intravesical Chronic Treatment Materials and Methods

Female 6-8 week old C57BL/6 mice were treated intravesically with eitherImiquimod (0.1 w/v % in total 208 nmol) or SC12 (0.38 w/v % in total206.5 nmol). Serum samples were taken at time points: day 0, 2 hours,day 1, 24 hours, and day 6, 2 hours.

Sample Preparation

Imiquimod: In a Sirocco Filter Plate (Waters), 50 microliters of mouseserum to 195 microliters of acetonitrile/methanol 1:1 spiked with 5microliters of IS (Imiquimod-D9 100 micrograms/ml).The plate was shakenfor 10 minutes and filtered under vacuum (5-10 mm Hg).

SC12: In a Sirocco Filter Plate (Waters), 70 microliters of mouse serumwere added to 210 microliters of acetonitrile/methanol 1:1 spiked with 5microliters of IS (Imiquimod-D9 100 ng/ml).The plate was shaken for 10minutes and filtered under vacuum (5-10 mm Hg). Samples were evaporatedand re-suspended in 70 microliters of acetonitrile/methanol 1:1.

Analytical Method

Imiquimod: LC/MS/MS: Premiere XE, Eluent: (ACN/H2O 95/5 (A)+0.1% HCOOH,5/95 (B)) flow 0.60 ml/min from 98% A 0-0.20 mins, gradient to 100% B in0.6 min, then stay to 100% B until 1.1 mins, reconditioning for 0.4 min.

Column: Acquity BEH C18 50×2.1 mm 1.7 micrometers; injection volume 5microliters, T column 50° C.

SC12: LC/MS/MS: Premiere XE, Eluent: (MeOH/H2O 95/5 (A)+0.1% HCOOH, 5/95(B)) flow 0.80 ml/min from 85% A 0-0.10 mins, gradient to 100% B in 0.4min, then stay to 100% B until 1.5 mins, reconditioning for 0.7 min.

Column: Acquity BEH C8 50×2.1 mm 1.7 micrometers injection. volume 10microliters, T column 50° C.

SC12 Q1/Q3 921.5/385.05; CV35, CE33 921.5/439.29; CV35, CE33 ImiquimodQ1/Q3 241.1/113.98; CV30, CE45 241.1/140.9; CV30, CE40 IS: Imiquimod-D9Q1/Q3 250.1/113.98; CV30, CE45 ESI positive, MRM, Extractor 5 V;Capillary 3.5 kV; T source 140° C.; T desolvation 450° C. LLOQ: 0.5ng/ml SC12 and 2.5 ng/ml for Imiquimod

Results

The aim of this study was to evaluate the exposure of serum afterintravesical chronic administration of Imiquimod and SC12. Serum levelsof SC12 and Imiquimod are reported in Table 33 and Table 34,respectively. Low concentrations were observed for both compounds, inparticular for SC12, where the major part of the samples were below theLLOQ, even if the LLOQ obtained for SC12 was five times lower than theLLOQ obtained for Imiquimod (0.5 vs 2.5 ng/ml). Imiquimod was present inthe serum up to 2 hours after administration, but no accumulation isoccurring resulting below the LLOQ at 24 hours and with values after 6days of treatment comparable to day 1.

TABLE 33 SC12 levels in serum Results are expressed as Mean ± S.D., n =2 Mice Day 0, 2 hours Day 1, 24 hours Day 6, 2 hours n^(o) ng/ml SC12, 91.0 <LLOQ <LLOQ 0.1% 10 <LLOQ <LLOQ <LLOQ 11 0.5 <LLOQ <LLOQ 12 <LLOQ<LLOQ <LLOQ Vehicle 13 <LLOQ <LLOQ <LLOQ Mean ± S.D. 0.5 ± 0.5 <LLOQ<LLOQ

TABLE 34 Imiquimod levels in serum Results are expressed as Mean ± S.D.,n = 2 Mice Day 0, 2 hours Day 1, 24 hours Day 6, 2 hours n^(o) ng/mlImiquimod 1 6.4 <LLOQ 8.7 0.1% 2 6.8 <LLOQ 3 5.3 <LLOQ 1 2.8 2 8.1 316.3 Vehicle 14 <LLOQ <LLOQ <LLOQ Mean ± S.D. 6.2 ± 0.8 <LLOQ 8.9 ± 5.6List of abbreviations Acetonitrile ACN Collision Energy CE Cone VoltageCV Dimethylsulfoxide DMSO Electron Spray Ionization ESI InternalStandard IS Liquid Chromatography/Mass Spectrometry LC-MS/MS Lowestlimit of quantification LLOQ Methanol MeOH Multiple Reaction MonitoringMRM Ultra Performance Liquid Chromatography UPLC

Example 17 Intracellular Uptake in RAW.264 Cells

In cellular assays, SC12 rapidly reaches high intracellularconcentration. 5×10⁶ RAW.264 cells were adhered overnight in 10 cmtissue culture dishes. The medium was replaced with 10 ml of a new mediacontaining 10 microM compound A and SC12. The cells were incubated for1, 6, and 18 hours. Supernatant (2 ml) and cells (pellets) werecollected by trypsinization and frozen at 20° C. for subsequent analysisby LC-MS. Table 35 shows the results of this analysis.

TABLE 35 Results of intracellular uptake assay Incubation IntracellularConcentration Compound hours % of total Compound A 1 5.50 6 4.27 16 1.53SC12 1 18.5 6 20.6 18 21.4

Example 18 Synthesis of SC14

The compound designated as tetradecanoic acid3-[(2-{4-[6-amino-2-(2-methoxy-ethoxy)-8-oxo-7,8-dihydro-purin-9-ylmethyl]-benzoylamino}-ethoxy)-hydroxy-phosphoryloxy]-2-tetradecanoyloxy-propylester (SC14) was synthesized as described below.

For preparing SC14, Acid 7 (compound 7) (Chimete, batch CH907/5/7b; 0.3g, 0.83 mmol) was dissolved in a mixture of dichloromethane anddimethylformamide (DMF) (9/1, 27 mL) at room temperature under an Argonatmosphere. Triethylamine (0.3 mL; 4 mmol) was added, followed by HATU(390 mg; 1.03 mmol). The resulting solution was stirred at roomtemperature for 10 min, followed by the addition of1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE; Avanti PolarLipids, Cas Number 998-07-2, 600 mg; 0.94 mmol). The solution was thenstirred overnight. The mixture was then diluted with water (100 mL). Asolid formed and was isolated by filtering with a Buchner funnelfollowed by washing with methanol (10 mL). SC14 was obtained as a paleyellow solid designated batch CH957/3/2 with a yield of 600 mg (73%) anda purity 95% as determined by HPLC. The structure was confirmed by¹H-NMR and MS.

Example 19 Synthesis of SC6

The SC6 designated as hexanoic acid3-[(2-{4-[6-amino-2-(2-methoxy-ethoxy)-8-oxo-7,8-dihydro-purin-9-ylmethyl]-benzoylamino}-ethoxy)-hydroxy-phosphoryloxy]-2-hexanoyloxy-propylester (SC6) was synthesized as described below.

For preparing SC6, Acid 7 (compound 7) (Chimete, batch CH907/5/7b; 0.5g, 1.39 mmol) was dissolved in a mixture of acetonitrile (10 mL) andDMSO (4 mL) at room temperature and under Argon atmosphere. Carbonyldiimidazole (Aldrich, batch 0001408017; 270 mg, 1.67 mmol, 1.05 eqv.)was added and the resulting solution was stirred for 1 hr. A solution of1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids;600 mg; 1.46 mmol.) was added in one portion. The reaction mixture wasstirred for 16 hours, until complete conversion of the reagents.Acetonitrile was removed by distillation in vacuum. Water (50 mL) wasadded to the residue and a white solid separated. The solid wasrecovered by centrifugation. The solid was purified by columnchromatography and was eluted with ethyl acetate/methanol/formic acid8/1/1. After drying in a vacuum at 35° C., SC6 was obtained as a whitesolid desinated batch CH957/2/1B with a yield of 500 mg (47%) and apurity of 96.4% as determined by HPLC. The structure was confirmed by1H-NMR and MS.

Example 20 Effect of SC14 on PBMC

To determine the effects of SC14 treatment on TNF-alpha and IL-6 releasefrom human PBMC (Peripheral Blood Mononuclear Cells), human PBMC wereisolated from buffy coats of human donors (Rho Hospital, Milan, Italy).Briefly, buffy coats from three healthy donors were diluted 1:1 insterile PBS (phosphate buffered saline). PBMCs were isolated by densitygradient centrifugation using Lymphocyte-H solution (CederlaneLaboratories). After two washing steps in PBS pH 7.4, cells wereresuspended in RPMI-1640, 10% FCS, L-glutamine (2×10⁴ M), penicillin(100 μml) and streptomycin (100 ug/ml). Three milliliters of a 1.6×10⁶ml suspension of PBMCs were seeded in six-well tissue culture plates.Wells were treated with either IMIQUIMOD, SC12, SC6 or SC14 atconcentrations of 50 uM, 10 uM, 2 uM, 0.4 uM, 0.08 uM and 0.016 uM. LPS(3 ul of a 1 mg/ml solution) was added to an untreated well as apositive control at a final concentration of 1 ug/ml. PBMCs were thenincubated for 24 hours. Supernatants were collected by centrifugation at1200 rpm for 10 minutes and frozen at −20° C. for later cytokineanalysis.

Cytokine concentrations were determined using a commercial Luminex kit(Fluorokine Map Multiplex Kit) for the simultaneous quantitativedetermination of multiple human cytokine concentrations. Mean TNF-alphaand IL-6 production levels after LPS stimulation are reported in Table36 as a positive control. TNF-alpha and IL-6 were detected at highlevels in the supernatant of PBMCs after treatment for 24 hours withSC12 or SC14, whereas IMIQUIMOD and SC6 did not show a significantstimulatory effect (Table 37 and FIG. 24 (TNF); Table 38 and FIG. 25(IL-6)).

All cytokine concentrations are presented as the Mean+/−SD (standarddeviation, n=2). In Table 37 and Table 38, an * indicates that one orboth values were extrapolated beyond the range of the standard. In Table38, OOR<indicates the data was out of range (i.e., below the range ofthe standard). Cytokine release was calculated per milliliter of plasmaand concentration units are pg/ml of medium.

TABLE 36 LPS Positive Control TNF-α IL-6 pg/ml SD pg/ml SD Donor 11167.5 42.2 *11837.9 329.2 Donor 2 802.7 47.4 *7613.8 516.4 Donor 3738.7 25.8 *11387.4 577.5

TABLE 37 TNF-alpha Release from Human PBMC TNF-α 0.016 μM 0.08 μM 0.4 μM2 μM 10 μM 50 μM pg/ml SD pg/ml SD pg/ml SD pg/ml SD pg/ml SD pg/ml SDDonor 1 IMIQUIMOD *3.48 0.20 *1.925 0.15 *0.93 0.01 15.29 0.21 31.982.11 11.75 0.36 SC12 *1.34 1.89 *4.89 0.47 173.46 25.69 1074.18 2.551471.62 442.53 *6680.36 124.32 SC-6 *1.60 0.01 1.38 0.01 *1.92 0.01*4.60 0.01 6.68 0.59 *5.64 0.30 SC-14 *2.25 0.01 41.37 0.01 189.47 13.17102.45 10.18 1244.23 7.00 1935.68 5.35 Donor 2 IMIQUIMOD *1.71 0.16*1.81 0.30 *1.49 0.47 6.88 0.53 22.36 0.63 11.91 0.71 SC12 *2.62 0.088.44 1.89 123.11 0.46 515.10 5.78 1250.81 102.97 3925.96 1264.79 SC-6*1.54 0.08 *1.10 0.08 *1.28 0.45 6.31 0.45 13.38 0.60 8.84 0.31 SC-14*4.90 0.22 59.86 2.66 197.06 28.09 559.85 339.62 1622.42 179.86 2785.56209.03 Donor 3 IMIQUIMOD *3.295 0.11 *2.43 0.23 *2.43 0.23 6.56 0.4220.04 0.30 12.52 1.41 SC12 *4.14 0.01 8.70 0.30 83.39 3.05 736.05 28.741939.86 147.50 *5377.97 653.40 SC-6 *2.59 0.45 *1.95 0.01 *3.83 1.308.91 0.01 7.15 0.42 9.36 0.21 SC-14 5.81 0.21 21.64 0.39 201.17 16.27783.56 22.06 2750.70 49.60 *5503.35 546.44

TABLE 38 IL-6 Release from Human PBMC IL-6 0.016 μM 0.08 μM 0.4 μM 2 μM10 μM 50 μM pg/ml SD pg/ml SD pg/ml SD pg/ml SD pg/ml SD pg/ml SD Donor1 IMIQUIMOD 23.09 2.02 *1.27 0.60 OOR< na 102.57 1.56 502.60 1.17 236.161.12 SC12 *3.51 0.32 23.37 0.85 1377.51 65.35 3987.04 21.12 4596.94106.06 *8902.80 113.99 SC-6 *0.59 0.36 OOR< — OOR< — 33.90 1.59 147.167.90 110.90 2.48 SC-14 *0.64 0.91 287.23 12.30 1475.62 101.17 788.6747.82 3634.59 114.22 *6308.86 24.37 Donor 2 IMIQUIMOD *1.07 0.31 *1.290.01 *0.99 1.00 17.05 1.60 164.50 8.42 189.53 7.83 SC12 *4.52 0.93 34.532.47 673.18 26.18 2118.17 265.54 3637.74 65.08 *5282.86 351.33 SC-6*2.47 0.01 *0.34 0.01 5.86 0.26 29.93 1.29 103.33 1.73 89.52 2.72 SC-1416.47 0.01 100.74 3.69 742.72 12.20 2910.82 43.96 5261.94 832.16*6404.48 668.12 Donor 3 IMIQUIMOD 18.62 0.66 6.84 0.84 6.62 1.67 149.3813.16 749.26 67.39 785.68 26.79 SC12 33.53 0.25 152.81 13.79 1377.2671.76 *5461.65 769.39 *7928.53 862.58 *10081.99 795.83 SC-6 9.99 0.278.13 0.42 19.54 2.76 137.54 2.60 145.17 8.68 173.86 18.22 SC-14 91.800.49 497.46 14.06 2829.73 124.13 *4542.78 33.15 *7825.40 649.58 *8462.40763.51

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A composition comprising a compound having a structure:


2. The composition of claim 1, which comprises a liposome.
 3. The composition of claim 1, which comprises an antigen.
 4. An immunostimulatory composition comprising a compound having a structure of:


5. The composition of claim 4, wherein the compound acts as an adjuvant.
 6. The composition of claim 4, which comprises an antigen.
 7. The composition of claim 4, which comprises a vaccine.
 8. A method for treating a condition in a subject, which comprises administering a composition of claim 1 to a subject in need thereof in an amount effective to treat the condition.
 9. A method for treating a condition in a subject, which comprises administering a composition of claim 4 to a subject in need thereof in an amount effective to treat the condition.
 10. A method for inducing an immune response in a subject, comprising administering to the subject a composition of claim 1 